Sequence and Structure-Based Analyses of Human Ankyrin Repeats

Ankyrin is one of the most abundant protein repeat families found across all forms of life. It is found in a variety of multi-domain and single domain proteins in humans with diverse number of repeating units. They are observed to occur in several functionally diverse proteins, such as transcriptional initiators, cell cycle regulators, cytoskeletal organizers, ion transporters, signal transducers, developmental regulators, and toxins, and, consequently, defects in ankyrin repeat proteins have been associated with a number of human diseases. In this study, we have classified the human ankyrin proteins into clusters based on the sequence similarity in their ankyrin repeat domains. We analyzed the amino acid compositional bias and consensus ankyrin motif sequence of the clusters to understand the diversity of the human ankyrin proteins. We carried out network-based structural analysis of human ankyrin proteins across different clusters and showed the association of conserved residues with topologically important residues identified by network centrality measures. The analysis of conserved and structurally important residues helps in understanding their role in structural stability and function of these proteins. In this paper, we also discuss the significance of these conserved residues in disease association across the human ankyrin protein clusters.

A symbiont phage protein aids in eukaryote immune evasion

Phages are increasingly recognized as important members of host associated microbial communities. While recent studies have revealed vast genomic diversity in the virosphere, the new frontier is to understand how newly discovered phages may affect higher order processes, such as in the context of host-microbe interactions. Here, we aim to understand the tripartite interplay between phages, bacterial symbionts and marine sponges. In a viromics approach, we discover 491 novel viral clusters and show that sponges, as filter-feeding organisms, are distinct viral niches. By using a nested sampling design, we show that each sponge individual of the four species investigated harbours its own unique virome, regardless of the tissue investigated. We further discover a novel, symbiont phage-encoded ankyrin domain-containing protein which appears to be widely spread in phages of many host-associated contexts including human. The ankyrin protein (ANKp) modulates the eukaryotic immune response against bacteria as confirmed in macrophage infection assays. We predict that the role of ANKp in nature is to facilitate co-existence in the tripartite interplay between phages, symbionts and sponges and possibly in many other host-microbe associations.

Ankyrin-Like Protein AnkB Interacts with CatB, Affects Catalase Activity, and Enhances Resistance ofXanthomonas oryzaepv. oryzae andXanthomonas oryzaepv. oryzicola to Phenazine-1-Carboxylic Acid

ABSTRACTXanthomonas oryzaepv. oryzae, which causes rice bacterial leaf blight, andXanthomonas oryzaepv. oryzicola, which causes rice bacterial leaf streak, are important plant-pathogenic bacteria. A member of the adaptor protein family, ankyrin protein, has been investigated largely in humans but rarely in plant-pathogenic bacteria. In this study, a novel ankyrin-like protein, AnkB, was identified inX. oryzaepv. oryzae andX. oryzaepv. oryzicola. The expression ofankBwas significantly upregulated when these bacteria were treated with phenazine-1-carboxylic acid (PCA).ankBis located 58 bp downstream of the genecatB(which encodes a catalase) in both bacteria, and the gene expression ofcatBand catalase activity were reduced followingankBdeletion inX. oryzaepv. oryzae andX. oryzaepv. oryzicola. Furthermore, we demonstrated that AnkB directly interacts with CatB by glutathioneS-transferase (GST) pulldown assays. Deletion ofankBincreased the sensitivity ofX. oryzaepv. oryzae andX. oryzaepv. oryzicola to H2O2and PCA, decreased bacterial biofilm formation, swimming ability, and exopolysaccharide (EPS) production, and also reduced virulence on rice. Together our results indicate that the ankyrin-like protein AnkB has important and conserved roles in antioxidant systems and pathogenicity inX. oryzaepv. oryzae andX. oryzaepv. oryzicola.IMPORTANCEThis study demonstrates that the ankyrin protein AnkB directly interacts with catalase CatB inXanthomonas oryzaepv. oryzae andXanthomonas oryzaepv. oryzicola. Ankyrin protein AnkB can affect the gene expression ofcatB, catalase activity, and sensitivity to H2O2. InXanthomonasspp., the locations of genesankBandcatBand the amino acid sequence of AnkB are highly conserved. It is suggested that in prokaryotes, AnkB plays a conserved role in the defense against oxidative stress.

Identification of adducin-binding residues on the cytoplasmic domain of erythrocyte membrane protein, band 3

Two major complexes form structural bridges that connect the erythrocyte membrane to its underlying spectrin-based cytoskeleton. Although the band 3–ankyrin bridge may account for most of the membrane-to-cytoskeleton interactions, the linkage between the cytoplasmic domain of band 3 (cdb3) and adducin has also been shown to be critical to membrane integrity. In the present paper, we demonstrate that adducin, a major component of the spectrin–actin junctional complex, binds primarily to residues 246–264 of cdb3, and mutation of two exposed glutamic acid residues within this sequence completely abrogates both α- and β-adducin binding. Because these residues are located next to the ankyrin-binding site on cdb3, it seems unlikely that band 3 can bind ankyrin and adducin concurrently, reducing the chances of an association between the ankyrin and junctional complexes that would significantly compromise erythrocyte membrane integrity. We also demonstrate that adducin binds the kidney isoform of cdb3, a spliceoform that lacks the first 65 amino acids of erythrocyte cdb3, including the central strand of a large β-pleated sheet. Because kidney cdb3 is not known to bind any of the common peripheral protein partners of erythrocyte cdb3, including ankyrin, protein 4.1, glyceraldehyde-3-phosphate dehydrogenase, aldolase, and phosphofructokinase, retention of this affinity for adducin was unexpected.

Molecular Characterization of Antibody Epitopes of Ehrlichia chaffeensis Ankyrin Protein 200 and Tandem Repeat Protein 47 and Evaluation of Synthetic Immunodeterminants for Serodiagnosis of Human Monocytotropic Ehrlichiosis

ABSTRACT Recently, major species-specific antibody epitopes in three immunoreactive tandem repeat proteins (TRPs) of Ehrlichia chaffeensis, TRP32, TRP47, and TRP120, have been identified and molecularly characterized within tandem repeat (TR) regions. In this study, we mapped the major immunodeterminants of the E. chaffeensis 200-kDa ankyrin protein (Ank200) and the minor immunodeterminants in the N- and C-terminal regions of E. chaffeensis TRP47. Major antibody epitopes of Ank200 were localized to four polypeptide regions (18-mer, 20-mer, 20-mer, and 21-mer, respectively) in terminal acidic domains, which reacted with antibodies in sera from human monocytotropic ehrlichiosis (HME) patients and an E. chaffeensis-infected dog. Two minor epitope-containing regions were identified in the N terminus and the C terminus of TRP47. The sensitivities and specificities of synthetic peptides representing these and other well-defined major immunodeterminants of E. chaffeensis were determined by enzyme-linked immunosorbent assay (ELISA). Thirty-one HME patient serum samples that had detectable E. chaffeensis antibodies (titers from 64 to 8,192) by indirect fluorescent-antibody assay (IFA) were tested. All 31 serum samples reacted with at least one E. chaffeensis peptide, 30 (96.8%) with TRP120 peptides, 27 (87.1%) with TRP32 peptides, 24 (77.4%) with TRP47 peptides, 19 (61.3%) with Ank200 peptides, and 28 (90.3%) with recombinant TRP120-TR protein. A mixture of the two most sensitive peptides from TRP120 and TRP32 did not provide enhanced analytical sensitivity compared to that provided by TRP120 alone. Our results demonstrate that the TRP120 peptide can be utilized for development of standardized sensitive point-of-care and reference laboratory immunodiagnostics for HME. This is the first study to compare analysis of molecularly defined major antibody epitopes with IFA for diagnosis of HME.