New Insights into the Function of N-Terminal 11 Amino Acids of Band 3 from Structural and Functional Study of a Naturally Occuring Band 3 Variant.

Abstract

The 911 amino acid human erythroid AE1 (eAE1) Cl-/HCO3- exchanger SLC4A1 (usually called band 3) is the major intrinsic membrane protein of red cells. The N-terminal cytoplasmic domain of AE1 represents the anchoring site for membrane-associated proteins such as ankyrin, protein 4.2, protein 4.1, glycolytic enzymes (including aldolase and glyceraldeyde-3-phosphate dehydrogenase (GAPDH) and hemoglobin. We identified marked band 3 deficiency in the second son of a consanguineous marriage with a life-threatening nonimmune hemolytic anemia. The patient was transfusion-dependent prior to splenectomy. SDS-PAGE and immunoblotting analysis of the proband red cell membrane proteins showed approximately 12±4% of band 3 and protein 4.2 compared to controls. Direct nucleotide sequence of SLC4A1 gene showed a single base substitution (T->C) at position +2 in the donor splice site of intron 2 (Band 3 Neapolis). Functionally, the mutation causes an altered splicing with the consequent formation of two different mature mRNAs, one including intron 2 and one skipping exon 2. While intron 2 retention leads to premature translation termination, exon 2 skipping causes the loss of the normal start site of eAE1 protein translation. The purification of mutant band 3 and its characterization by MALDI mass spectrometry demonstrated the lack of the first 11 amino acids due to the usage of second in frame start site. Real-time RT-PCR analyses of reticulocyte mRNA showed a marked decrement in band 3 transcription accounting for protein deficiency. The lack of the 11 N-terminal amino acids resulted in complete absence of membrane bound aldolase while other glycolitic enzymes (for example GAPDH) were membrane bound. Syk tyrosine kinase recognized the truncated band 3 as a substrate in vitro. In spite of this ability to be phosphorylated by Syk and to recruit Lyn tyrosine kinase in vitro, we were unable to demonstrate Tyr-phosphorylation of mutant band 3 in intact erythrocytes following stimulation by oxidative stress. This finding implies a requirement for the 11 N-terminal amino acids for the sequential Tyr-phosphorylation of band 3 in intact red cell membranes. The mutant band 3 was largely present in the high molecular weight aggregate fraction (about 5.2 fold higher than control), indicating its increased tendency to cluster in the membrane. The spontaneous clustering of truncated band 3 strongly suggests that the negatively charged N-terminal domain may regulate oligomeric state of band 3 in the membrane. Biophysical characterization showed that band 3 deficiency resulted in decreased cohesion between lipid bilyer and spectrin based membrane skeleton accounting for membrane loss. The structural and functional characterization of the naturally occuring mutant band 3 has enabled us to identify a significant role for the 11 N-terminal amino acids in band 3 function and in red cell membrane physiology.