Characterization of a thermoacidophilic L-arabinose isomerase from Alicyclobacillus acidocaldarius: role of Lys-269 in pH optimum

The araA gene encoding L-arabinose isomerase (AI) from the thermoacidophilic bacterium Alicyclobacillus acidocaldarius was cloned, sequenced, and expressed in Escherichia coli. Analysis of the sequence revealed that the open reading frame of the araA gene consists of 1,491 bp that encodes a protein of 497 amino acid residues with a calculated molecular mass of 56,043 Da. Comparison of the deduced amino acid sequence of A. acidocaldarius AI (AAAI) with other AIs demonstrated that AAAI has 97% and 66% identities (99% and 83% similarities) to Geobacillus stearothermophilus AI (GSAI) and Bacillus halodurans AI (BHAI), respectively. The recombinant AAAI was purified to homogeneity by heat treatment, ion-exchange chromatography, and gel filtration. The purified enzyme showed maximal activity at pH 6.0 to 6.5 and 65 degrees C under the assay conditions used, and it required divalent cations such as Mn2+, Co2+, and Mg2+ for its activity. The isoelectric point (pI) of the enzyme was about 5.0 (calculated pI of 5.5). The apparent Km values of the recombinant AAAI for L-arabinose and D-galactose were 48.0 mM (Vmax, 35.5 U/mg) and 129 mM (Vmax, 7.5 U/mg), respectively, at pH 6 and 65 degrees C. Interestingly, although the biochemical properties of AAAI are quite similar to those of GSAI and BHAI, the three AIs from A. acidocaldarius (pH 6), G. stearothermophilus (pH 7), and B. halodurans (pH 8) exhibited different pH activity profiles. Based on alignment of the amino acid sequences of these homologous AIs, we propose that the Lys-269 residue of AAAI may be responsible for the ability of the enzyme to act at low pH. To verify the role of Lys-269, we prepared the mutants AAAI-K269E and BHAI-E268K by site-directed mutagenesis and compared their kinetic parameters with those of wild-type AIs at various pHs. The pH optima of both AAAI-K269E and BHAI-E268K were rendered by 1.0 units (pH 6 to 7 and 8 to 7, respectively) compared to the wild-type enzymes. In addition, the catalytic efficiency (kcat/Km) of each mutant at different pHs was significantly affected by an increase or decrease in Vmax. From these results, we propose that the position corresponding to the Lys-269 residue of AAAI could play an important role in the determination of the pH optima of homologous AIs.     

The role of post-translational modifications of the CXCR4 amino terminus in stromal-derived factor 1 alpha association and HIV-1 entry

The chemokine receptor CXCR4 plays critical roles in development, immune function, and human immunodeficiency virus type 1 (HIV-1) entry. Here we demonstrate that, like the CC-chemokine receptors CCR5 and CCR2b, CXCR4 is posttranslationally modified by sulfation of its amino-terminal tyrosines. The sulfate group at tyrosine 21 contributes substantially to the ability of CXCR4 to bind its ligand, stromal derived factor 1 alpha. Tyrosine sulfation plays a less significant role in CXCR4-dependent HIV-1 entry than in CCR5-dependent HIV-1 entry. In some cell lines, CXCR4 is efficiently modified by a chondroitin sulfate chain at serine 18, but neither HIV-1 entry nor stromal derived factor 1 alpha binding was affected by loss of this glycosaminoglycan. These data demonstrate a functional role for tyrosine sulfate in the CXC-chemokine receptor family and underscore a general difference in HIV-1 utilization of CCR5 and CXCR4.     

Morphological and Molecular Identification of Spirometra Tapeworms (Cestoda: Diphyllobothriidae) from Carnivorous Mammals in the Serengeti and Selous Ecosystems of Tanzania

Spirometra tapeworms (Cestoda: Diphyllobothriidae) collected from carnivorous mammals in Tanzania were identified by the DNA sequence analysis of the mitochondrial cytochrome c oxidase subunit 1 (cox1) and internal transcribed spacer 1 (ITS1), and by morphological characteristics. A total of 15 adult worms were collected from stool samples and carcasses of Panthera leo, Panthera pardus, and Crocuta crocuta in the Serengeti and Selous ecosystems of Tanzania. Three Spirometra species: S. theileri, S. ranarum and S. erinaceieuropaei were identified based on morphological features. Partial cox1 sequences (400 bp) of 10 specimens were revealed. Eight specimens showed 99.5% similarity with Spirometra theileri ({"type":"entrez-nucleotide","attrs":{"text":"MK955901","term_id":"1796114604","term_text":"MK955901"}}MK955901), 1 specimen showed 99.5% similarity with the Korean S. erinaceieuropaei and 1 specimen had 99.5% similarity with Myanmar S. ranarum. Sequence homology estimates for the ITS1 region of S. theileri were 89.8% with S. erinaceieuropaei, 82.5% with S. decipiens, and 78.3% with S. ranarum; and 94.4% homology was observed between S. decipiens and S. ranarum. Phylogenetic analyses were performed with 4 species of Spirometra and 2 species of Dibothriocephalus (=Diphyllobothrium). By both ML and BI methods, cox1 and ITS1 gave well supported, congruent trees topology of S. erinaceieuropaei and S. theileri with S. decipiens and S. ranarum forming a clade. The Dibothriocephalus species were sisters of each other and collectively forming successive outgroups. Our findings confirmed that 3 Spirometra species (S. theileri, S. ranarum, and S. erinaceieuropaei) are distributed in the Serengeti and Selous ecosystems of Tanzania.     

A functional interaction between chromogranin B and the inositol 1,4,5-trisphosphate receptor/Ca2+ channel

Chromogranins A and B (CGA and CGB) are high capacity, low affinity calcium (Ca2+) storage proteins found in many cell types most often associated with secretory granules of secretory cells but also with the endoplasmic reticulum (ER) lumen of these cells. Both CGA and CGB associate with inositol 1,4,5-trisphosphate receptor (InsP3R) in a pH-dependent manner. At an intraluminal pH of 5.5, as found in secretory vesicles, both CGA and CGB bind to the InsP3R. When the intraluminal pH is 7.5, as found in the ER, CGA totally dissociates from InsP3R, whereas CGB only partially dissociates. To investigate the functional consequences of the interaction between the InsP3R and CGB monomers or CGA/CGB heteromers, purified mouse InsP3R type I were fused to planar lipid bilayers and activated by 2 microM InsP3. In the presence of luminal CGB monomers or CGA/CGB heteromers the InsP3R/Ca2+ channel open probability and mean open time increased significantly. The channel activity remained elevated when the pH was changed to 7.5, a reflection of CGB binding to the InsP3R even at pH 7.5. These results suggest that CGB may play an important modulatory role in the control of Ca2+ release from the ER. Furthermore, the difference in the ability of CGA and CGB to regulate the InsP3R/Ca2+ channel and the variability of CGA/CGB ratios could influence the pattern of InsP3-mediated Ca2+ release.     

A tyrosine-sulfated CCR5-mimetic peptide promotes conformational transitions in the HIV-1 envelope glycoprotein

The HIV-1 envelope glycoprotein is a trimeric complex of heterodimers composed of a surface glycoprotein, gp120, and a transmembrane component, gp41. The association of this complex with CD4 stabilizes the coreceptor-binding site of gp120 and promotes the exposure of the gp41 helical region 1 (HR1). Here, we show that a 15-amino-acid peptide mimetic of the HIV-1 coreceptor CCR5 fused to a dimeric antibody Fc domain (CCR5mim-Ig) bound two gp120 molecules per envelope glycoprotein complex and by itself promoted HR1 exposure. CCR5mim-Ig also stabilized the association of a CD4-mimetic peptide with the envelope glycoprotein. A fusion of the CD4- and CCR5-mimetic peptides, DM1, bound gp120 and neutralized R5, R5X4, and X4 HIV-1 isolates comparably to CD4, and they did so markedly more efficiently than either peptide alone. Our data indicate that the potency of DM1-Ig derives from its avidity for the HIV-1 envelope glycoprotein trimer and from the bidirectional induction of its receptor-mimetic components. DM1 has significant advantages over other inhibitors that target both coreceptor and CD4-binding sites, and it may serve as a lead for a new class of HIV-1 inhibitor peptides.     

An in vitro airway wall model of remodeling

Recent studies have shown that mechanical forces on airway epithelial cells can induce upregulation of genes involved in airway remodeling in diseases such as asthma. However, the relevance of these responses to airway wall remodeling is still unclear since 1). mechanotransduction is highly dependent on environment (e.g., matrix and other cell types) and 2). inflammatory mediators, which strongly affect remodeling, are also present in asthma. To assess the effects of mechanical forces on the airway wall in a relevant three-dimensional inflammatory context, we have established a tissue culture model of the human airway wall that can be induced to undergo matrix remodeling. Our model contains differentiated human bronchial epithelial cells characterized by tight junctions, cilia formation, and mucus secretion atop a collagen gel embedded with human lung fibroblasts. We found that addition of activated eosinophils and the application of 50% strain to the same system increased the epithelial thickness compared with either condition alone, suggesting that mechanical strain affects airway wall remodeling synergistically with inflammation. This integrated model more closely mimics airway wall remodeling than single-cell, conditioned media, or even two-dimensional coculture systems and is relevant for examining the importance of mechanical strain on airway wall remodeling in an inflammatory environment, which may be crucial for understanding and treating pathologies such as asthma.     

Identification of a binding site on the type II activin receptor for activin and inhibin

Type II activin receptors (ActRII and ActRIIB) are single-transmembrane domain serine/threonine kinase receptors that bind activin to initiate the signaling and cellular responses triggered by this hormone. Inhibin also binds type II activin receptors and antagonizes many activin effects. Here we describe alanine scanning mutagenesis of the ActRII extracellular domain. We identify a cluster of three hydrophobic residues (Phe(42), Trp(60), and Phe(83)) that, when individually mutated to alanine in the context of the full-length receptor, cause the disruption of activin and inhibin binding to ActRII. Each of the alanine-substituted ActRII mutants retaining activin binding maintains the ability to form cross-linked complexes with activin and supports activin cross-linking to the type I activin receptor ALK4. Unlike wild-type ActRII, the three mutants unable to bind activin do not cause an increase in activin signaling when transiently expressed in a corticotroph cell line. Together, our results implicate these residues in forming a critical binding surface on ActRII required for functional interactions with both activin and inhibin. This first identification of a transforming growth factor-beta family member binding site may provide a general basis for characterizing binding sites for other members of the superfamily.