HUMAN ERYTHROID BAND-3 ANION-EXCHANGER-1 IS EXPRESSED IN TRANSFORMED LYMPHOCYTES

The anion transporter, band 3, is a ubiquitous protein. It is present in brain and all other tissues examined. Not only is band 3 present in cell membranes, but also in nuclear, Golgi and mitochondria membranes . There are four isoforms of band 3, the anion exchanger (AE) proteins , thus far discovered. They are products of different genes. Lymphocyt es are reported to contain AE2, but not AE1. We hypothesized that indu ction or up-regulation of AE1 occurs when lymphocytes are transformed as an initial event in the path to malignancy. We transformed lymphocy tes containing a single base mutation with Epstein Barr Virus (EBV). T he mutation of band 3, high transport band 3 (HTbd3), exhibits anion t ransport that is 2-3 times normal in erythrocytes which contain AE1. T his facilitated our identification of AE1 since the probability that 2 different gene products would have the same mutation approaches zero. Thus, we have a base mutation in addition to linear sequence to ident ify AE1. A 133 base pair (bp) fragment including the affected region w as amplified from the mRNA of lymphocytes from the HTbd3 mutant. AE1 p rimers were used to amplify regions of interest. Reverse transcriptase polymerase chain reaction (RT-PCR) was used to generate cDNA which wa s sequenced. The sequence of the crucial 133 base pair segment from hi gh transport lymphoctes was 100% identical to the sequence published f or the red blood cell band 3 of the same mutant. As reported previousl y for erythrocytes, this mutation is a C-->T base change which changes a proline to leucine in the protein sequence. Restriction enzyme dige sts of AE1 cDNA from normal and HTBD3 lymphocytes confirmed that the p roposita was homozygous for the mutation, and showed the father to be heterozygous. Anion transport was increased in HTbd3 EBV transformed l ymphocytes, as was the case with HTbd3 erythrocytes. AE2 was identifie d in lymphocytes by sequence. Thus,:EBV transformed lymphocytes expres s the erythroid band 3 (AE1) in addition to AE2.