Michael J. A. Tanner

The structure and function of band 3 (AE1): Recent developments (Review)

This review discusses recent advances in our understanding of the structure, function and molecular genetics of the membrane domain of red cell anion exchanger, band 3 (AE1), and its role in red cell and kidney disease. A new model for the topology of band 3 has been proposed, which suggests the membrane domain has 12 membrane spans, rather than the 14 membrane spans of earlier models. The major difference between the models is in the topology of the region on the C-terminal side of membrane spans 1-7. Two dimensional crystals of the deglycosylated membrane domain of band 3 have yielded two and three dimensional projection maps of the membrane domain dimer at low resolution. The human band 3 gene has been completely sequenced and this has facilitated the study of natural band 3 mutations and their involvement in disease. About 20% of hereditary spherocytosis cases arise from heterozygosity for band 3 mutations, and result in the absence or decrease of the mutant protein in the red cell membrane. Several other natural band 3 mutations are known that appear to be clinically benign, but alter red cell phenotype or are associated with altered red cell blood group antigens. These include the mutant band 3 present in Southeast Asian ovalocytosis, a condition which provides protection against cerebral malaria in children. Familial distal renal tubular acidosis, a condition associated with kidney stones, has been shown to result from a novel group of band 3 mutations. The total absence of band 3 has been described in animals-occurring naturally in cattle and after targeted disruption in mice. Some of these severely anaemic animals survive, so band 3 is not strictly essential for life. Although the band 3-negative red cells were very unstable, they contained a normally-assembled red cell skeleton, suggesting that the bilayer of the normal red cell membrane is stabilized by band 3 interactions with membrane lipids, rather than by interactions with the spectrin skeleton.

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 α-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 μ1B-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.

Basis of unique red cell membrane properties in hereditary ovalocytosis

Hereditary ovalocytes from a Mauritian subject are extremely rigid, with a shear elastic modulus about three times that of normal cells, and have increased resistance to invasion by the malaria parasite Plasmodium falciparum in vitro. The genetic anomaly resides in band 3; the protein gives rise to chymotryptic fragments with reduced mobility in SDS/polyacrylamide gel electrophoresis, but this is a result of anomalous binding of SDS and not a higher molecular weight. Analysis of the band 3 gene reveals (1) a point mutation (Lys56----Glu), which also occurs in a common asymptomatic band 3 (Memphis) variant and governs the electrophoretic properties, and (2) a deletion of nine amino acid residues, including a proline residue, encompassing the interface between the membrane-associated and the N-terminal cytoplasmic domains. The interaction of the mutant band 3 with ankyrin appears unperturbed. The fraction of band 3 capable of undergoing translation diffusion in the membrane is greatly reduced in the ovalocytes. Cells containing the asymptomatic band 3 variant were normal with respect to all the properties that we have studied. Possible mechanisms by which a structural change in band 3 at the membrane interface could regulate rigidity are examined.

Biochemical aspects of the blood group Rh (rhesus) antigens.

Despite their importance in clinical haematology, the details of the structures and possible functions of the proteins associated with Rh antigen expression have only recently begun to emerge. The antigens are carried by a multimeric complex between a M(r) 30,000 polypeptide which is not glycosylated (the Rh30 polypeptide), and a heavily glycosylated glycoprotein (the Rh50 glycoprotein). The N-terminal amino acid sequences of the two types of proteins were determined and used to isolated cDNA clones. The Rh30 and Rh50 proteins are both very hydrophobic membrane proteins, each containing up to 12 membrane spans. The two proteins are homologous in sequence and clearly belong to the same family. They are erythroid-specific and not related to any other known family of proteins. The Rh30 polypeptides are the genetic determinants of Rh blood group antigen activity. One polypeptide (Rh30A) is probably associated with CcEe antigen activity, while another (Rh30B) is responsible for the D antigen. The proteins have structures typical of transporters but their functions are still unclear. A number of other red cell membrane proteins (LW, CD47, glycophorin B and Fy) show alterations in red cells lacking Rh antigens (Rhnull). These proteins may have a role in the biosynthesis or function of the Rh30 and Rh50 proteins.

Reducible components in the proteins of human erythrocyte membrane.

In contrast to a previous report, no collagen or elastin-type cross-linked derived from lysine-aldehydes were detected in human erythrocyte membranes. The major reducible components of erythrocyte membranes were shown to be hexosyllysines. From their structure it is clear that these components cannot act as cross-links between the protein subunits of the membrane. The components were also shown to be present in varying proportions in human serum albumin and haemoglobin. Whether the hexose attachments have any physiological significance or are artefacts of the analytical procedure has not yet been demonstrated. One other major reducible component was present but, although unidentified, this compound was shown to be unrelated to any of the known lysine-aldehyde-derived cross-links of collagen and elastin. A minor acidic component was identified as glucosylvaline derived from the N-terminus of the beta chain of haemoglobin A1c and not a lysine-aldehyde precursor of the collagen cross-links.

The Rhesus (D) polypeptide is linked to the human erythrocyte cytoskeleton

Cytoskeleton preparations derived from lactoperoxidase‐radioiodinated human erythrocytes were found to be enriched in a labelled component with the same apparent molecular mass as the Rhesus (D) (Rh(D)) antigen polypeptide. Immune precipitation from the cytoskeleton preparations confirmed that this component is the Rh(D) polypeptide. The results suggest that the Rh(D) polypeptide may be linked to the erythrocyte skeletal matrix. The possibility that the Rh(D) antigen is involved in maintaining the shape and viability of the erythrocyte is discussed.

Incomplete glycosylation of erythrocyte membrane proteins in congenital dyserythropoietic anaemia Type II (CDA II)

The alterations in the erythrocyte membrane proteins of individuals with congenital dyserythropoietic anaemia (CDA II) were studied. Alterations were observed in both the erythrocyte sialoglycoproteins and erythrocyte anion transport protein (Band 3). There was a decrease in the apparent molecular weight of the major sialoglycoprotein α (glycophorin A) as well as a general reduction in the intensity of staining of all the sialoglycoproteins by the PAS stain. Sialoglycoprotein α isolated from CDA II erythrocytes contained 30% less sialic acid than normal α. The anion transport protein of CDA II erythrocytes migrated as a band with a lower molecular weight than the normal protein on SDS‐gel electrophoresis. The CDA II anion transport protein had a substantially reduced content of N‐acetylglucosamine and galactose, which probably reflects a reduction in the number of N‐acetyl‐lactosamine units carried by the protein. Our results suggest that there is a general defect in glycosylation of the major membrane glycoproteins of CDA II erythrocytes. We suggest that this glycosylation defect is a consequence of bone marrow stress.

South-east Asian ovalocytic (SAO) erythrocytes have a cold sensitive cation leak: Implications for in vitro studies on stored SAO red cells

South-east Asian ovalocytosis (SAO) results from the heterozygous presence of an abnormal band 3, which causes several alterations in the properties of the erythrocytes. Although earlier studies suggested that SAO erythrocytes are refractory to invasion in vitro by the malarial parasite Plasmodium falciparum, a more recent study showed that fresh SAO cells were invaded by the parasites, but became resistant to invasion on storage because intracellular ATP was depleted more rapidly than normal. Here we show that SAO red cells are much more leaky to sodium and potassium than normal red cells when stored in the cold. This leak was much less marked when the cells were stored at 25 or 37 degreesC. Incubation for 3.5 h at 37 degreesC of cold-stored SAO red cells did not restore sodium and potassium to normal levels, probably because the depleted ATP level in cold-stored SAO red cells is further reduced with incubation at 37 degreesC. The increased leakiness of SAO red cells is non-specific and extends to calcium ions, taurine, mannitol and sucrose. These results suggest that SAO red cells undergo a structural change on cooling. Since many of the reports describing altered properties of SAO red cells have used cells which have been stored in the cold, these results need re-evaluation using never-chilled SAO red cells to assess whether the cells have the same abnormal properties under in vivo conditions.

Role of N-glycosylation in the expression of human band 3-mediated anion transport

The human erythrocyte anion transporter (band 3; AE1) has a single N-linked glycosylation site at amino residue Asn-642. To investigate the functional role of the N-glycan in band 3 (b3) we have constructed mutant b3 cDNAs in which this residue has been replaced by Gly, Ser or Thr, and the expression of these mutants was examined in Xenopus oocytes. Chymotrypsin treatment of intact oocytes was used to assess surface b3. Similar amounts of cleavage were observed with both glycosylated and unglycosylated b3. Greater cleavage of b3 was obtained when human red cell glycophorin A (GPA) was co-expressed with either glycosylated or unglycosylated b3. The co-expression of GPA with either glycosylated or unglycosylated b3 increased the stilbene disulphonate-sensitive chloride transport into oocytes at low cRNA concentrations. In both the presence or absence of GPA, a higher b3-mediated chloride influx into oocytes was observed on expression of glycosylated b3 cRNA compared with similar amounts of unglycosylated b3 cRNA. We suggest that glycosylation is not essential for the expression of functional b3 in oocytes, but may play a role in enabling the protein to acquire its correct folding with the highest anion transport activity.

Synthesis of a branched cyclic peptide using a strategy employing Fmoc chemistry and two additional orthogonal protecting groups

Abstract A novel branched cyclic peptide has been made by solid-phase synthesis (Fmoc/t-Bu chemistry) using two additional orthogonal strategies (Allyl and Dde) and resin-bound cyclization.