- Email: [email protected]
Functional cell surface expression of band 3, the human red blood cell anion exchange protein (AE1), in K562 erythroleukemia cells: Band 3 enhances the cell surface reactivity of Rh antigens
242
Molecular basis of weak D phenotypes. Wagner FF, Gassner C, MOiler TH, et aL Blood 93:385-393, 1999. Previous publications=from at least four laboratories have reported a normal RHD coding sequence in weak D samples. In contrast, this article showed that all of 161 weak D samples studied had an altered RHD exon sequence. The samples were from blood donors in southwestern Germany identified by nonreactivity with anti-D in direct agglutination tests and reactivity in the indirect antiglobulin test. Partial D v[ samples were excluded from the analysis. Seqnencing of nucleotides in stretches of genomic DNA representative of all l0 RHD exons and parts of the promoter region was accomplished using 14 primers specific for RHD and 26 primers specific for RHD or P(HCE in different combinations of primers used for polymerase chain reaction (PCR) amplification and for sequencing. This approach eliminated the need for subcloning. With PCRrestriction fragment length polymorphisms (PCR-RFLP) and PCR-sequence-specific priming (PCR-SSP) methods, 18 distinct RHD alleles encoding amino acid changes were identified. One was D TM type III (RHD-CE [exon 6-9]-D) and one DHMi. Of the remaining 16 patterns, 14 had single, different, previously unknown missense mutations, one had two nucleotide changes (type 4), and one had three nucleotide changes (type 14) typical for the RHCE gene interspersed in the RHD-specific sequences. All of the amino acid substitutions were located in the predicted intracellular or transmembraneous domains of RhD. The authors propose that these weak D types be referred to by trivial names, for example, weak D type 1, or by their molecular structures, such as RHD (V270G). The weak D type 1 was the most common (95 of 161), with a prevalence of 1 in 277, and in all cases was associated with the R~ haplotype. Weak D type 2 was the next most common (43 of 161), with a prevalence of 1 in 1,082, and in all cases was associated with the R2 haplotype. The other 14 weak D types were present in seven or fewer donors. The amino acid substitutions occurred along the RhD protein from residues 3 to 393. Seven of the substitutions were between residues 270 and 307. The authors discuss possible exPlanations as to why other groups have not found these point mutations in blood with a weak expression of the D antigen. The findings described in this article provide information about the amino acids involved in optimal insertion of RhD into the RBC membrane. Amino acids at positions 295 and 385 may be particularly important. %vo alleles (associated with type 4 and type 14) had nucleotides in exons 4 and 5 substituted by the equivalent nucleotides from the RHCE gene. Similar (although more extensive) exchanges occur in D vl type I and D w type II, which also have reduced expression of RhD. Furthermore, the results provide a possible explanation for paradoxical observations. For example, Dma,iwhich differs from weak D type 4 only by an N152T substitution, has a normal D antigen density, suggesting that the N152 T substitution in exon 3 may facilitate the membrane integration. In addition, DInc, DIV% and D vI type III each harbor the N152T substitution and have enhanced RhD densities when compared with normal D and D vI type II, respectively. Experimental proof of this hypothesis awaits development of a suitable expression system. This raises the question that if N152 is important for full expression of RhD, why is it not equally important for expression of the highly homologous RhCE protein, which has thronine at protein 152? (M.E.R.)
CURRENT LITERATURE
Functional cell surface expression of band 3, the human red blood cell anion exchange protein (AE1), in K562 erythro!eukemia cells: Band 3 enhances the cell surface reactivity of Rh antigens. Beckmann R, Smythe JS, Anstee DJ, et al. Blood 92:4428-4438, 1998. Human erythroleukemia cells (K562) were transfected with human band 3 cDNA using the pBabe retroviral vector as previously reported by these investigators. Stable clones expressing band 3 were isolated by flow cytometry. Expression of band 3, measured by monoclonal anti-band 3 (BRIC 6), was ninefold in the K562/B3 clone as compared with the K562 clone transfected with empty pBp vector (K562/pBp). Tw9 monoclonal anti-GPA .antibodies (R10 and R18) bound strongly to both KK562/B3 and K562/pBp clones. The antibodies bound slightly more strongly to the K562/pBp clone cells than to the control cells. Anti-Wrb (BRIC 14 and BRIC 201) showed, respectively, a 42-fold and an eightfold increase in binding. There was a threefold increase in binding of monoclonal antibodies (MAbs) to RhAG (LA18.18) and LW (BS46) glycoproteins. Antibodies to an intracellular epitope of Band 3 (BRIC 169), to GPC (BRIC 4), and to Fy3 (CBC-512 and to CD47 (BRIC 126) gave only slight differences in binding, and this was attributed to the high affinity of K562/B3 clone for the secondary antibody. Light microscopy could not distinguish K562 cells expressing band 3 from K562 or K562/pBp cells. However, by flow cytometry, the K562/Band 3 cells were slightly more heterogenous in cell size and granularity. The function of the band 3 expressed at the surface of transfected K562 cells was demonstrated by chloride transport assays. The authors quantitated the amount of band 3. expressed by density analysis of immunoblots. The density of cell surface band 3 molecules is estimated to be 50 times greater in RBCs than in the K562/B3 cells. Coexpression of K562 clones that bad previously been transduced with cDNA encoding either RhD or RhcE with band 3 cDNA substantially increased the levels of RhD and RhcE antigen expression. There was also increased expression of the endogenous Rh glycoprotein. The authors suggest that the increased reactivity of Rh antigens may result from cell surfade or intracellular interactions of band 3 with the protein complex that contains the Rh proteins and RhAG, or from indirect effects of band 3 on the membrane environment. All clones that expressed band 3 also expressed high levels of Wr b antigen, which was not expressed on untransfected cells or K562/pBp cells. This showed that the expressed band 3 and the endogenous GPA in K562 cells are able to interact as they do in RBCs and provides direct evidence that band 3 is required for formation of the Wr b epitopes. (M.E.R.)
The Fy x phenotype is associated with a missense mutation in the Fyb allele predicting Arg89Cys in the Duffy glycoprotein. O/sson ML, Smythe JS, Hansson C, eta/. BrJ Haemato1103:1184-1191, 1998. The Duffy glycoprotein is a receptor f ~ chemokines and for the malarial parasites Plasmodium vivax and P knowlesi. The molecular basis for the Fy~/Fyb polymorphism is caused by a missense mutation resulting in a single amino acid difference of Gly42Asp (numbering based on the major red blood cell Duffy protein). The molecular event responsible for the inactivation of the silent allele among blacks is due to a single-nucleotide substitution (T to C) in the FY promoter region, thereby
Molecular basis of weak D phenotypes. Wagner FF, Gassner C, MOiler TH, et aL Blood 93:385-393, 1999. Previous publications=from at least four laboratories have reported a normal RHD coding sequence in weak D samples. In contrast, this article showed that all of 161 weak D samples studied had an altered RHD exon sequence. The samples were from blood donors in southwestern Germany identified by nonreactivity with anti-D in direct agglutination tests and reactivity in the indirect antiglobulin test. Partial D v[ samples were excluded from the analysis. Seqnencing of nucleotides in stretches of genomic DNA representative of all l0 RHD exons and parts of the promoter region was accomplished using 14 primers specific for RHD and 26 primers specific for RHD or P(HCE in different combinations of primers used for polymerase chain reaction (PCR) amplification and for sequencing. This approach eliminated the need for subcloning. With PCRrestriction fragment length polymorphisms (PCR-RFLP) and PCR-sequence-specific priming (PCR-SSP) methods, 18 distinct RHD alleles encoding amino acid changes were identified. One was D TM type III (RHD-CE [exon 6-9]-D) and one DHMi. Of the remaining 16 patterns, 14 had single, different, previously unknown missense mutations, one had two nucleotide changes (type 4), and one had three nucleotide changes (type 14) typical for the RHCE gene interspersed in the RHD-specific sequences. All of the amino acid substitutions were located in the predicted intracellular or transmembraneous domains of RhD. The authors propose that these weak D types be referred to by trivial names, for example, weak D type 1, or by their molecular structures, such as RHD (V270G). The weak D type 1 was the most common (95 of 161), with a prevalence of 1 in 277, and in all cases was associated with the R~ haplotype. Weak D type 2 was the next most common (43 of 161), with a prevalence of 1 in 1,082, and in all cases was associated with the R2 haplotype. The other 14 weak D types were present in seven or fewer donors. The amino acid substitutions occurred along the RhD protein from residues 3 to 393. Seven of the substitutions were between residues 270 and 307. The authors discuss possible exPlanations as to why other groups have not found these point mutations in blood with a weak expression of the D antigen. The findings described in this article provide information about the amino acids involved in optimal insertion of RhD into the RBC membrane. Amino acids at positions 295 and 385 may be particularly important. %vo alleles (associated with type 4 and type 14) had nucleotides in exons 4 and 5 substituted by the equivalent nucleotides from the RHCE gene. Similar (although more extensive) exchanges occur in D vl type I and D w type II, which also have reduced expression of RhD. Furthermore, the results provide a possible explanation for paradoxical observations. For example, Dma,iwhich differs from weak D type 4 only by an N152T substitution, has a normal D antigen density, suggesting that the N152 T substitution in exon 3 may facilitate the membrane integration. In addition, DInc, DIV% and D vI type III each harbor the N152T substitution and have enhanced RhD densities when compared with normal D and D vI type II, respectively. Experimental proof of this hypothesis awaits development of a suitable expression system. This raises the question that if N152 is important for full expression of RhD, why is it not equally important for expression of the highly homologous RhCE protein, which has thronine at protein 152? (M.E.R.)
CURRENT LITERATURE
Functional cell surface expression of band 3, the human red blood cell anion exchange protein (AE1), in K562 erythro!eukemia cells: Band 3 enhances the cell surface reactivity of Rh antigens. Beckmann R, Smythe JS, Anstee DJ, et al. Blood 92:4428-4438, 1998. Human erythroleukemia cells (K562) were transfected with human band 3 cDNA using the pBabe retroviral vector as previously reported by these investigators. Stable clones expressing band 3 were isolated by flow cytometry. Expression of band 3, measured by monoclonal anti-band 3 (BRIC 6), was ninefold in the K562/B3 clone as compared with the K562 clone transfected with empty pBp vector (K562/pBp). Tw9 monoclonal anti-GPA .antibodies (R10 and R18) bound strongly to both KK562/B3 and K562/pBp clones. The antibodies bound slightly more strongly to the K562/pBp clone cells than to the control cells. Anti-Wrb (BRIC 14 and BRIC 201) showed, respectively, a 42-fold and an eightfold increase in binding. There was a threefold increase in binding of monoclonal antibodies (MAbs) to RhAG (LA18.18) and LW (BS46) glycoproteins. Antibodies to an intracellular epitope of Band 3 (BRIC 169), to GPC (BRIC 4), and to Fy3 (CBC-512 and to CD47 (BRIC 126) gave only slight differences in binding, and this was attributed to the high affinity of K562/B3 clone for the secondary antibody. Light microscopy could not distinguish K562 cells expressing band 3 from K562 or K562/pBp cells. However, by flow cytometry, the K562/Band 3 cells were slightly more heterogenous in cell size and granularity. The function of the band 3 expressed at the surface of transfected K562 cells was demonstrated by chloride transport assays. The authors quantitated the amount of band 3. expressed by density analysis of immunoblots. The density of cell surface band 3 molecules is estimated to be 50 times greater in RBCs than in the K562/B3 cells. Coexpression of K562 clones that bad previously been transduced with cDNA encoding either RhD or RhcE with band 3 cDNA substantially increased the levels of RhD and RhcE antigen expression. There was also increased expression of the endogenous Rh glycoprotein. The authors suggest that the increased reactivity of Rh antigens may result from cell surfade or intracellular interactions of band 3 with the protein complex that contains the Rh proteins and RhAG, or from indirect effects of band 3 on the membrane environment. All clones that expressed band 3 also expressed high levels of Wr b antigen, which was not expressed on untransfected cells or K562/pBp cells. This showed that the expressed band 3 and the endogenous GPA in K562 cells are able to interact as they do in RBCs and provides direct evidence that band 3 is required for formation of the Wr b epitopes. (M.E.R.)
The Fy x phenotype is associated with a missense mutation in the Fyb allele predicting Arg89Cys in the Duffy glycoprotein. O/sson ML, Smythe JS, Hansson C, eta/. BrJ Haemato1103:1184-1191, 1998. The Duffy glycoprotein is a receptor f ~ chemokines and for the malarial parasites Plasmodium vivax and P knowlesi. The molecular basis for the Fy~/Fyb polymorphism is caused by a missense mutation resulting in a single amino acid difference of Gly42Asp (numbering based on the major red blood cell Duffy protein). The molecular event responsible for the inactivation of the silent allele among blacks is due to a single-nucleotide substitution (T to C) in the FY promoter region, thereby