Identification of contact sites between ankyrin and band 3 in the human erythrocyte membrane.

The red cell membrane is stabilized by a spectrin/actin-based cortical cytoskeleton connected to the phospholipid bilayer via multiple protein bridges. By virtue of its interaction with ankyrin and adducin, the anion transporter, band 3 (AE1), contributes prominently to these bridges. In a previous study, we demonstrated that an exposed loop comprising residues 175-185 of the cytoplasmic domain of band 3 (cdB3) constitutes a critical docking site for ankyrin on band 3. In this paper, we demonstrate that an adjacent loop, comprising residues 63-73 of cdB3, is also essential for ankyrin binding. Data that support this hypothesis include the following. (1) Deletion or mutation of residues within the latter loop abrogates ankyrin binding without affecting cdB3 structure or its other functions. (2) Association of cdB3 with ankyrin is inhibited by competition with the loop peptide. (3) Resealing of the loop peptide into erythrocyte ghosts alters membrane morphology and stability. To characterize cdB3-ankyrin interaction further, we identified their interfacial contact sites using molecular docking software and the crystal structures of D(3)D(4)-ankyrin and cdB3. The best fit for the interaction reveals multiple salt bridges and hydrophobic contacts between the two proteins. The most important ion pair interactions are (i) cdB3 K69-ankyrin E645, (ii) cdB3 E72-ankyrin K611, and (iii) cdB3 D183-ankyrin N601 and Q634. Mutation of these four residues on ankyrin yielded an ankyrin with a native CD spectrum but little or no affinity for cdB3. These data define the docking interface between cdB3 and ankyrin in greater detail.

Regulation of membrane-cytoskeletal interactions by tyrosine phosphorylation of erythrocyte band 3.

The cytoplasmic domain of band 3 serves as a center of erythrocyte membrane organization and constitutes the major substrate of erythrocyte tyrosine kinases. Tyrosine phosphorylation of band 3 is induced by several physiologic stimuli, including malaria parasite invasion, cell shrinkage, normal cell aging, and oxidant stress (thalassemias, sickle cell disease, glucose-6-phosphate dehydrogenase deficiency, etc). In an effort to characterize the biologic sequelae of band 3 tyrosine phosphorylation, we looked for changes in the polypeptide's function that accompany its phosphorylation. We report that tyrosine phosphorylation promotes dissociation of band 3 from the spectrin-actin skeleton as evidenced by: (1) a decrease in ankyrin affinity in direct binding studies, (2) an increase in detergent extractability of band 3 from ghosts, (3) a rise in band 3 cross-linkability by bis-sulfosuccinimidyl-suberate, (4) significant changes in erythrocyte morphology, and (5) elevation of the rate of band 3 diffusion in intact cells. Because release of band 3 from its ankyrin and adducin linkages to the cytoskeleton can facilitate changes in multiple membrane properties, tyrosine phosphorylation of band 3 is argued to enable adaptive changes in erythrocyte biology that permit the cell to respond to the above stresses.

Mutations in the phenylalanine hydroxylase gene identified in 95 patients with phenylketonuria using novel systems of mutation scanning and specific genotyping based upon thermal melt profiles.

Phenylketonuria (PKU, MIM 261600; EC 1.14.16.1) results from mutations in the phenylalanine hydroxylase (PAH) gene. Newborn metabolic disease screening uses blood dried on filter paper (DBS) to prospectively identify candidate newborns affected with PKU via an elevated concentration of phenylalanine. However, it is then important to confirm the specific category of PKU since classical PKU requires a stringent diet while milder categories may not require diet and a very important BH4-responsive category may be treated with the PAH cofactor 6R-tetrahydrobiopterin (BH4). Since there is a close genotype-phenotype correlation in PKU, determining the PAH genotype can be extremely important for therapy as well as prognosis. A simple and rapid method of accurately determining the PAH genotype would be a valuable addition to the diagnosis of PKU. Described herein is a means to identify variants in the PAH gene using high-resolution melt profiling, which compares the thermal denaturation profile of a patient sample to that of a control. Regions where the patient and control samples produce a common profile were not further evaluated, while those regions where the patient profile deviates from the control were assessed by DNA sequencing. Additionally described is a scheme utilizing redundant analysis with melt profile controls and a novel multiplex genotyping assay to triage deviation owing to known polymorphisms. Two mutations were identified in 93 of the 95 patients assessed and in the remaining two patients a single mutation was identified. Melt profiling provided 99% sensitivity to identify sequence variants in the PAH gene.