Isolation and characterization of poly-N-acetyllactosaminylceramides accumulated in the erythrocytes of congenital dyserythropoietic anemia type II patients

Congenital dyserythropoietic anemia type II or hereditary erythroblastic polynuclearity with positive acidified serum test (HEMPAS) is a rare genetic disease inherited by a recessive mode. Previous studies on HEMPAS erythrocytes have shown that Band 3 and Band 4.5 glycoproteins were not glycosylated by lactosaminoglycans, while polylactosaminyl carbohydrates are accumulated as glycolipids (P. Scartezzini et al., Br J. Haematol., 51 (1982) 569; M.N. Fukuda et al., Br. J. Haematol., 56 (1984)55). Presently, we have isolated polylactosaminyl lipids from HEMPAS blood cells and analyzed their structures by fast atom bombardment-mass spectrometry (FAB-MS), methylation analysis, endo-beta-galactosidase digestion. The results indicate that polylactosaminyl lipids accumulated in HEMPAS erythrocytes are a species of poly-N-acetyllactosaminylceramides which are also present in normal erythrocytes, but at 7 approximately 9 times lower level. Isolated polylactosaminylceramides exhibit I-, i-, H- and Lex antigenic activities which suggest that the polylactosaminylceramides are derived from both erythrocytes and granulocytes.     

HEMPAS disease: genetic defect of glycosylation

Congenital dyserythropoietic anaemia Type II or HEMPAS (hereditary erythroblastic multinuclearity with positive acidified serum lysis test) is a rare genetic anaemia in humans, inherited in an autosomally recessive mode. Biochemical analyses of HEMPAS erythrocyte membranes suggested strongly that HEMPAS is caused by defective glycosylation of erythrocyte membrane glycoproteins. Most recently a HEMPAS case has been identified as being defective in the gene encoding Golgi alpha-mannosidase II by using cDNA probe of alpha-mannosidase II. At present, it is not clear whether HEMPAS is a genetically heterogenous collection of glycosylation deficiencies, as some HEMPAS cases showed a low level of N-acetylglucosaminyltransferase II. Abnormal glycosylation of serum glycoproteins and association of liver cirrhosis in HEMPAS patients indicate that HEMPAS disease is not restricted to erythroid cells. On the other hand, normal development of HEMPAS patients during embryonic stage strongly suggests the possibilities of fetal type isozyme in place of defective glycosylation enzyme.     

HEMPAS. Hereditary erythroblastic multinuclearity with positive acidified serum lysis test

Congenital dyserythropoietic anemia type II or HEMPAS (hereditary erythroblastic multinuclearity with positive acidified serum lysis test) is a genetic anemia in humans caused by a glycosylation deficiency. Erythrocyte membrane glycoproteins, such as band 3 and band 4.5, which are normally glycosylated with polylactosamines lack these carbohydrates in HEMPAS. Polylactosamines accumulate as glycolipids in HEMPAS erythrocytes. Analysis of N-glycans from HEMPAS erythrocyte membranes revealed a series of incompletely processed N-glycan structures, indicating defective glycosylation at N-acetylglucosaminyltransferase II (GnT-II) and/or alpha-mannosidase II (MII) steps. Genetic analysis has identified two cases from England in which the MII gene is defective. Mutant mice in which the MII gene was inactivated by homologous recombination resulted in a HEMPAS-like phenotype. On the other hand, linkage analysis of HEMPAS cases from southern Italy excluded MII and GnT-II as the causative gene, but identified a gene on chromosome 20q11. HEMPAS is therefore genetically heterogeneous. Regardless of which gene is defective, HEMPAS is characterized by incomplete processing of N-glycans. The study of HEMPAS will identify hitherto unknown factors affecting N-glycan synthesis.     

Primary defect of congenital dyserythropoietic anemia type II. Failure in glycosylation of erythrocyte lactosaminoglycan proteins caused by lowered N-acetylglucosaminyltransferase II

Congenital dyserythropoietic anemia type II or hereditary erythroblastic multinuclearity with positive acidified serum test (HEMPAS) is a genetic disease caused by membrane abnormality. Previously we have found that Band 3 and Band 4.5 are not glycosylated by lactosaminoglycans in HEMPAS erythrocytes, whereas normally these proteins have lactosaminoglycans (Fukuda, M. N., Papayannopoulou, T., Gordon-Smith, E. C., Rochant, H., and Testa, U. (1984) Br. J. Haematol. 56, 55-68). In order to find out where glycosylation of lactosaminoglycans stops, we have analyzed the carbohydrate structures of HEMPAS Band 3. By fast atom bombardment-mass spectrometry, methylation analysis, and hydrazinolysis followed by exoglycosidase treatments, the following structure was elucidated: (formula; see text) N-Linked glycopeptides synthesized in vitro by reticulocyte microsomes from HEMPAS were shown to be predominantly the above short oligosaccharide, whereas those from normal reticulocytes contain large molecular weight carbohydrates. The N-acetylglucosaminyltransferase II, which transfers N-acetylglucosamine to the C-2 position of the Man alpha 1----6Man beta 1----arm of the biantennary core structure, was therefore examined by using Man alpha 1----6(GlcNAc beta 1----2Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4GlcNAcol as an acceptor. N-Acetylglucosaminyltransferase II activity was demonstrated in the lymphocyte microsome fraction from normal individuals. However, this enzyme activity was found to be decreased in those from HEMPAS patients. These results suggest that the primary defect of HEMPAS lies in the lowered activity of N-acetylglucosaminyltransferase II.     

Polylactosamines are not obligate receptors for invasion of Plasmodium falciparum malaria as shown in HEMPAS Variant II-gal- erythrocytes

A HEMPAS (hereditary erythroblastic multinuclearity with positiveacidified serum test) erythrocyte, atypical Variant II (referredto herein as Variant II-gal^-), lacking long-chain polylactosamineon both glycoproteins (Band 3 and 4.5) and glycosphingolipids,was characterized by the carbohydrate profile of the erythrocytemembrane according to Fukuda et al. (Blood, 73, 1331–1339,1989). Two laboratories previously reported that polylactosamineisolated from the erythrocyte protein Band 3 inhibited invasionof red blood cells by Plasmodium falciparum in malarial culture,suggesting a role for this carbohydrate in adhesion of the parasite.Therefore, HEMPAS erythrocyte Variant II-gal^- presented a uniqueopportunity to further examine this premise. Freshly drawn bloodsamples (normal and HEMPAS Variant II-gal^-) were separatelyincubated with P.falciparum from mannitol-synchronized cultures.The parasite was found to invade HEMPAS Variant II-gal^- erythrocytesat a 30% lower rate through two life cycles, as shown by microscopicevaluation of invasion and by [³H]hypoxanthine incorporationinto parasite. This observation, along with the published factthat glycophorin-deficient M^kM^k cells are also infectable, butat a lower rate, indicates that neither sialoglycoproteins norpolylactosamines are an obligate adhesive ligand for P.falciparum,although the possibility remains that either may still contributeto adhesive events during infection. HEMPAS malaria Plasmodium falciparum polylactosamine     

The association between familial distal renal tubular acidosis and mutations in the red cell anion exchanger (band 3, AE1) gene.

In distal renal tubular acidosis (dRTA) the tubular secretion of hydrogen ion in the distal nephron is impaired, leading to the development of metabolic acidosis, frequently accompanied by hypokalemia, nephrocalcinosis, and metabolic bone disease. The condition can be familial, when it is usually inherited as an autosomal dominant, though there is a rarer autosomal recessive form associated with nerve deafness. It has been shown that the autosomal dominant form of dRTA is associated with a defect in the anion exchanger (AE1) of the renal collecting duct intercalated cell. This transporter is a product of the same gene (AE1) as the erythrocyte anion exchanger, band 3. In this review we will look at the evidence for this association. Studies of genomic DNA from families with this disorder have shown, both by genetic linkage studies and by DNA sequencing, that affected individuals are heterozygous for mutations in the AE1 gene whilst unaffected family members have a normal band 3 sequence. Mutations have been found in the region of proposed helices 6 and 7 of the membrane domain of band 3 and involve amino acids Arg-589 and Ser-613, and in the COOH-terminal domain of band 3. Studies of red cell band 3 from these families have provided information on the effect these mutations have on the structure and function of erythrocyte band 3. Expression studies of the erythroid and kidney isoforms of the mutant AE1 proteins, in Xenopus laevis oocytes, have shown that they retained chloride transport activity, suggesting that the disease in the dRTA families is not related simply to the anion transport activity of the mutated proteins. A possible explanation for the dominant effect of these mutant AE1 proteins in the kidney cell is that these mutations affect the targeting of AE1 from the basolateral to the apical membrane of the alpha-intercalated cell.     

Genetic tailoring of N-linked oligosaccharides: the role of glucose residues in glycoprotein processing of Saccharomyces cerevisiae in vivo

In higher eukaryotes a quality control system monitoring the folding state of glycoproteins is located in the ER and is composed of the proteins calnexin, calreticulin, glucosidase II, and UDP-glucose: glycoprotein glucosyltransferase. It is believed that the innermost glucose residue of the N- linked oligosaccharide of a glycoprotein serves as a tag in this control system and therefore performs an important function in the protein folding pathway. To address this function, we constructed Saccharomyces cerevisiae strains which contain nonglucosylated (G0), monoglucosylated (G1), or diglucosylated (G2) glycoproteins in the ER and used these strains to study the role of glucose residues in the ER processing of glycoproteins. These alterations of the oligosaccharide structure did not result in a growth phenotype, but the induction of the unfolded protein response upon treatment with DTT was much higher in G0 and G2 strains as compared to wild-type and G1 strains. Our results provide in vivo evidence that the G1 oligosaccharide is an active oligosaccharide structure in the ER glycoprotein processing pathway of S.cerevisiae. Furthermore, by analyzing N- linked oligosaccharides of the constructed strains we can directly show that no general glycoprotein glucosyltransferase exists in S. cerevisiae.      

Occurrence of O-glycosidically peptide-linked oligosaccharides of poly-N-acetyllactosamine type (erythroglycan II) in the I-antigenically active Sendai virus receptor sialoglycoprotein GP-2

Unique high molecular weight (M.W. 4,000-9,000) sugar chains termed erythroglycan II have been obtained from alkali/sodium borohydride digests of I-active asialoglycoprotein derived from sialoglycoprotein GP-2, which was isolated recently from bovine erythrocyte membranes as Sendai virus receptor (Suzuki, Y. et al. (1983) J. Biochem. 93, 1621-1633; (1984) ibid, 95, 1193-1200). It was found that these sugar chains comprise about 40% of total alkali-labile oligosaccharides of asialo GP-2 and contain endo-beta-galactosidase (Flavobacterium keratolyticus)-resistant highly branched and heterogeneous oligosaccharides of poly-N-acetyllactosamine type which are linked O-glycosidically to the peptide backbone through N-acetylgalactosamine. Erythroglycan II also contains endo-beta-galactosidase-susceptible straight terminal polylactosaminyl side chains. A major oligosaccharide released by the enzyme cochromatographed with Gal beta 1-4GlcNAc beta 1-3Gal. Inhibitory activity of Sendai virus-mediated hemagglutination and the receptor activity for the virus were reduced significantly but not completely by the endo-beta-galactosidase. These results indicate that both linear and branched sialosylpolylactosamine sequences in erythroglycan II are important for the reception of the virus into the target cells.     

Role of the nucleoside transport function in the transport and salvage of purine nucleobases

Genetic deficiencies in the nucleoside transport function markedly altered the abilities of cultured mutant S49 T lymphoblasts to transport, incorporate, and salvage exogenous hypoxanthine. The concentrations of exogenous hypoxanthine required to reverse azaserine toxicity and replenish azaserine-depleted nucleoside triphosphate pools in AE1 cells, a nucleoside transport-deficient clone, were about 10-fold higher than those required for wild type cells. In a similar fashion, guanine could reverse mycophenolic acid toxicity in wild type but not in AE1 cells. Surprisingly, a second nucleoside transport-deficient clone, 80-5D2, which had lost 80-90% of its ability to transport nucleosides, required lower hypoxanthine concentrations than the wild type parent to reverse these azaserine-mediated effects. The addition of submicromolar concentrations of either p-nitrobenzylthioinosine or dipyridamole, two potent inhibitors of nucleoside transport, to wild type cells mimicked the phenotype of the AE1 cells with respect to hypoxanthine. AE1 cells or p-nitrobenzylthioinosine-treated wild type cells could only transport hypoxanthine at 10-25% the rate of untreated wild type cells, whereas 80-5D2 cells could transport hypoxanthine more efficiently. Adenine transport was also diminished in AE1 and FURD-80-3-6 cells, but not to sufficiently low levels to interfere with their ability to salvage adenine to overcome azaserine toxicity. These studies on S49 cells altered in their nucleoside transport capacity provide powerful genetic evidence that purine nucleobases share a common transport function with nucleosides in these mammalian T lymphoblasts.     

Sequence Determinants of GLUT1 Oligomerization: ANALYSIS BY HOMOLOGY-SCANNING MUTAGENESIS*

The human blood-brain barrier glucose transport protein (GLUT1) forms homodimers and homotetramers in detergent micelles and in cell membranes, where the GLUT1 oligomeric state determines GLUT1 transport behavior. GLUT1 and the neuronal glucose transporter GLUT3 do not form heterocomplexes in human embryonic kidney 293 (HEK293) cells as judged by co-immunoprecipitation assays. Using homology-scanning mutagenesis in which GLUT1 domains are substituted with equivalent GLUT3 domains and vice versa, we show that GLUT1 transmembrane helix 9 (TM9) is necessary for optimal association of GLUT1-GLUT3 chimeras with parental GLUT1 in HEK cells. GLUT1 TMs 2, 5, 8, and 11 also contribute to a less abundant heterocomplex. Cell surface GLUT1 and GLUT3 containing GLUT1 TM9 are 4-fold more catalytically active than GLUT3 and GLUT1 containing GLUT3 TM9. GLUT1 and GLUT3 display allosteric transport behavior. Size exclusion chromatography of detergent solubilized, purified GLUT1 resolves GLUT1/lipid/detergent micelles as 6- and 10-nm Stokes radius particles, which correspond to GLUT1 dimers and tetramers, respectively. Studies with GLUTs expressed in and solubilized from HEK cells show that HEK cell GLUT1 resolves as 6- and 10-nm Stokes radius particles, whereas GLUT3 resolves as a 6-nm particle. Substitution of GLUT3 TM9 with GLUT1 TM9 causes chimeric GLUT3 to resolve as 6- and 10-nm Stokes radius particles. Substitution of GLUT1 TM9 with GLUT3 TM9 causes chimeric GLUT1 to resolve as a mixture of 6- and 4-nm particles. We discuss these findings in the context of determinants of GLUT oligomeric structure and transport function.     

Recycling cell surface glycoproteins undergo limited oligosaccharide reprocessing in LEC1 mutant Chinese hamster ovary cells

The ability of particular cell surface glycoproteins to recycle and become exposed to individual Golgi enzymes has been demonstrated. This study was designed to determine whether endocytic trafficking includes significant reentry into the overall oligosaccharide processing pathway. The Lec1 mutant of Chinese hamster ovary (CHO) cells lack N - acetylglucosaminyltransferase I (GlcNAc-TI) activity resulting in surface expression of incompletely processed Man5GlcNAc2 N -linked oligosaccharides. An oligosaccharide tracer was created by exoglycosylation of cell surface glycoproteins with purified porcine GlcNAc-TI and UDP-[3H]GlcNAc. Upon reculturing, all cell surface glycoproteins that acquired [3H]GlcNAc were acted upon by intracellular mannosidase II, the next enzyme in the Golgi processing pathway of complex N -linked oligosaccharides (t1/2= 3-4 h). That all radiolabeled cell surface glycoproteins were included in this endocytic pathway indicates a common intracellular compartment into which endocytosed cell surface glycoproteins return. Significantly, no evidence was found for continued oligosaccharide processing consistent with transit through the latter cisternae of the Golgi apparatus. These data indicate that, although recycling plasma membrane glycoproteins can be reexposed to individual Golgi-derived enzymes, significant reentry into the overall contiguous processing pathway is not evident.      

Studies on the effect of glycoprotein processing inhibitors on fusion of L6 myoblast cell lines

The effect of oligosaccharide processing inhibitors on the fusion of L6 myoblasts was studied. The glucosidase inhibitors, castanospermine, 1-deoxynojirimycin and N-methyl-deoxynojirimycin were potent inhibitors of myoblast fusion, as was the mannosidase II inhibitor, swainsonine. Inhibition of fusion was reversed when inhibitors were removed. However, the mannosidase I inhibitor, 1-deoxymannojirimycin did not inhibit fusion. Changes in cell membrane oligosaccharide structure were followed by monitoring the binding of concanavalin A (conA) and wheat germ agglutinin (WGA) to cell surface membranes in cells treated with processing inhibitors. All the processing inhibitors resulted in increased binding of conA and decreased binding of WGA; this is consistent with the known mechanisms of inhibition of the inhibitors used in the study. Inhibition of fusion by the processing inhibitors also resulted in reduced activities of creatine phosphokinase, an enzyme used as a marker for biochemical differentiation during fusion. Treatment of a non-differentiating conA-resistant cell line with processing inhibitors did not induce fusion, but the cells did show altered lectin-binding properties. The main conclusion drawn from these studies is that cell surface glycoproteins probably containing the mannose (Man)9 structure are important for the fusion reaction.     

Interaction of glucose transporter 1 with anion exchanger 1 in vitro

The facilitative glucose transporter 1 (GLUT1) mediates the passive diffusion of d-glucose across the cell membrane, providing the energy resource in glycolysis in the erythrocytes. Anion exchanger 1 (band 3) is another important membrane protein that mediates rapid exchange of CO(2) through Cl(-)/HCO(3)(-) exchange across the erythrocyte membrane. For verifying the presumption over a decade that GLUT1 and band 3 in the erythrocyte would be interacting with each other, we cloned and expressed both the cytoplasmic domains of GLUT1 and band 3 in Escherichia coli, and tested their binding ability. By coimmunoprecipitation we found that among the tested N-terminal, C-terminal, and loop fraction of GLUT1, only the C-terminal of GLUT1 can interact with cytoplasmic domain of band 3. The interaction was further verified by coimmunoprecipitation and pull-down assay using both proteins as bait and target. These results showed that GLUT1 and band 3 form a protein complex that can regulate the activities of the proteins within it.     

Structure of an unusual complex-type oligosaccharide isolated from Chinese hamster ovary cells

An asparagine-linked oligosaccharide with an unusual structure has been isolated from Pronase digests of Chinese hamster ovary cell glycoproteins using gel filtration, ion exchange chromatography, and affinity chromatography on lectin-Sepharose columns. The oligosaccharide contained approximately 7.5% of the total cellular glycopeptide galactose and 1.3% of the mannose. The structure of the oligosaccharide was determined by the combination of methylation analysis and digestion with exo- and endo-glycosidases. The oligosaccharide has predominantly a triantennary structure consisting of repeating [Galβ1 → 4GlcNAcβ1 → 3] units in the outer branches linked to a trimannosyl-di-N,N′-acetyl-chitobiose core. It resembles oligosaccharides present on human erythrocyte glycoproteins and corneal keratan sulfate.     

The membrane change in En(a-) human erythrocytes. Absence of the major erythrocyte sialoglycoprotein.

We investigated the membrane of En(a-) human erythrocytes as part of a study of the structure and biochemical function of the surface glycoproteins of the mammalian cell. 2. En(a-) erythrocytes were selected because they have more extensive changes at the cell surface than any other known erythrocyte variant. 3. Our results show that in En(a-) erythrocytes: (a) the major membrane sialoglycoprotein is lacking; (b) the other major membrane-penetrating glycoprotein (band 3) has an altered electrophoretic mobility. 4. The apparent clinical normality of En(a-) cells suggests that the change in band 3 may compensate for the loss of the membrane sialoglycoproteins. It is clear that a viable erythrocyte can exist despite the absence of one of its major surface components.     

Addition of truncated oligosaccharides to influenza virus hemagglutinin results in its temperature-conditional cell-surface expression

In the preceding paper (Hearing, J., E. Hunter, L. Rodgers, M.-J. Gething, and J. Sambrook. 1989. J. Cell Biol. 108:339-353) we described the isolation and initial characterization of seven Chinese hamster ovary cell lines that are temperature conditional for the cell-surface expression of influenza virus hemagglutinin (HA) and other integral membrane glycoproteins. Two of these cell lines appeared to be defective for the synthesis and/or addition of mannose-rich oligosaccharide chains to nascent glycoproteins. In this paper we show that at both 32 and 39 degrees C in two mutant cell lines accumulate a truncated version, Man5GlcNAc2, of the normal lipid-linked precursor oligosaccharide, Glc3Man9GlcNAc2. This is possibly due to a defect in the synthesis of dolichol phosphate because in vitro assays indicate that the mutant cells are not deficient in mannosylphosphoryldolichol synthase at either temperature. A mixture of truncated and complete oligosaccharide chains was transferred to newly synthesized glycoproteins at both the permissive and restrictive temperatures. Both mutant cell lines exhibited altered sensitivity to cytotoxic plant lectins when grown at 32 degrees C, indicating that cellular glycoproteins bearing abnormal oligosaccharide chains were transported to the cell surface at the permissive temperature. Although glycosylation was defective at both 32 and 39 degrees C, the cell lines were temperature conditional for growth, suggesting that cellular glycoproteins were adversely affected by the glycosylation defect at the elevated temperature. The temperature-conditional expression of HA on the cell surface was shown to be due to impairment at 39 degrees C of the folding, trimerization, and stability of HA molecules containing truncated oligosaccharide chains.     

Glycosidase inhibitors: inhibitors of N-linked oligosaccharide processing.

The biosynthesis of the various types of N-linked oligosaccharide structures involves two series of reactions: 1) the formation of the lipid-linked saccharide precursor, Glc3Man9(GlcNAc)2-pyrophosphoryl-dolichol, by the stepwise addition of GlcNAc, mannose and glucose to dolichyl-P, and 2) the removal of glucose and mannose by membrane-bound glycosidases and the addition of GlcNAc, galactose, sialic acid, and fucose by Golgi-localized glycosyltransferases to produce different complex oligosaccharide structures. For most glycoproteins, the precise role of the carbohydrate is still not known, but specific N-linked oligosaccharide structures are key players in targeting of lysosomal hydrolases to the lysosomes, in the clearance of asialoglycoproteins from the serum, and in some cases of cell:cell adhesion. Furthermore, many glycoproteins have more than one N-linked oligosaccharide, and these oligosaccharides on the same protein frequently have different structures. Thus, one oligosaccharide may be of the high-mannose type whereas another may be a complex chain. One approach to determining the role of specific structures in glycoprotein function is to use inhibitors that block the modification reactions at different steps, causing the cell to produce glycoproteins with altered carbohydrate structures. The function of these glycoproteins can then be assessed. A number of alkaloid-like compounds have been identified that are specific inhibitors of the glucosidases and mannosidases involved in glycoprotein processing. These compounds cause the formation of glycoproteins with glucose-containing high mannose structures, or various high-mannose or hybrid chains, depending on the site of inhibition. These inhibitors have also been useful for studying the processing pathway and for comparing processing enzymes from different organisms.     

alpha-Glucosidase II-deficient cells use endo alpha-mannosidase as a bypass route for N-linked oligosaccharide processing.

The kinetics of N-linked oligosaccharide processing and the structures of the processing intermediates have been examined in normal parental BW5147 mouse lymphoma cells and the alpha-glucosidase II-deficient PHAR2.7 mutant cells. The mutant cells accumulated glucosylated intermediates but were able to deglucosylate and process about 40% of their oligosaccharides to complex-type. This processing was not due to residual alpha-glucosidase II activity since the alpha-glucosidase inhibitors 1-deoxynojirimycin (DNJ) and N-butyl-DNJ did not prevent it. Parent cells also showed alpha-glucosidase II-independent processing in the presence of DNJ and N-butyl-DNJ. Membrane preparations from both parent and mutant cells had endo alpha-mannosidase activity, that is, split Glc1,2Man9GlcNAc to Glc1,2Man plus Man8GlcNAc, indicating that this was a candidate for an alternate route to complex oligosaccharide formation in the mutant cells. A balance study in which the cellular glycoproteins, intracellular water soluble saccharides, and saccharides secreted into the medium were isolated and analyzed from [2-3H]mannose-labeled mutant cells showed that the cells formed the di- and trisaccharides Glc1Man and Glc2Man in amounts equivalent to the deglucosylated oligosaccharides found in the cellular glycoproteins. This result shows unequivocally that the alpha-glucosidase II-deficient mutant cells use endo alpha-mannosidase as a bypass route for N-linked oligosaccharide processing.     

Integral membrane protein interaction with Triton cytoskeletons of erythrocytes

The organization of erythrocyte membrane lipids and proteins has been studied following the release of cytoplasmic components with the non-ionic detergent Triton X-100. After detergent extraction, a detergent-resistant complex called the erythrocyte cytoskeleton is separated from detergent, solubilized lipid and protein by sucrose buoyant density sedimentation. In cytoskeletons prepared under isotonic conditions all of the major erythrocyte membrane proteins are retained except for the integral protein, glycophorin, which is quantitatively solubilized and another integral glycoprotein, band 3, which is only 60% removed. When cytoskeletons are prepared in hypertonic KCl solutions, band 3 is fully solubilized along with bands 2.1 and 4.2 and several minor components. The resulting cytoskeletons have the same morphology as those prepared in isotonic buffer but they are composed of only three major peripheral proteins, spectrin, actin and band 4.1. We have designated this peripheral protein complex the 'shell' of the erythrocyte membrane, and have shown that the attachment of band 3 to the shell satisfies the criteria for a specific interaction. Although Triton did affect erythrocyte shape, cytoskeleton lipid content and the activity of membrane proteases, there was no indication that Triton altered the attachment of band 3 to the shell. We suggest that band 3 attaches to the shell as part of a ternary complex of bands 2.1, 3 and 4.2.     

Assessment of erythrocyte deformability with the laser-assisted optical rotational cell analyzer (LORCA)

The erythrocyte deformability of 28 patients with anemia was evaluated with the laser-assisted optical rotational cell analyzer (LORCA), an image analyzer that converts into numerical form the degree of refraction of a laser beam induced by red cells subjected to a range of torsional stresses. The patients were 10 thalassemics, including three with intermediate forms (1 HbC/beta degree, 1 homozygote beta for Orkin's haplotype VI, 1 beta degree/beta delta Sicilian type) and seven heteroygotes for beta Th; six with hereditary spherocytosis (including 2 with structural alteration of the spectrin beta chain); three with type II congenital dyserythropoietic anemia (HEMPAS), two hemizygotes and one heterozygote for G-6PD deficiency, and six with severe hypochromic hyposideremic anemia. Red cell deformability was reduced in intermediate thalassemia, hereditary spherocytosis and HEMPAS, normal in heterozygous beta thalassemia and G-6PD deficiency, and increased in hypochromic hyposideremic anemia. These results show that erythrocyte deformability can be impaired by an Hb chain imbalance, membrane and cyto skeleton structure anomalies and changes in the red cell area/volume ratio.