Myxococcus xanthus twin-arginine translocation system is important for growth and development

The twin-arginine translocation (Tat) system serves to export fully folded proteins across the cytoplasmic membrane. In many bacteria, three major components, TatA, TatB and TatC, are the functionally essential constituents of the Tat system. A Myxococcus xanthus tatB–tatC deletion mutant could aggregate and form mounds, but was unable to form fruiting bodies under nutritionally limiting conditions. When tatB–tatC mutant vegetative cells were cultured with 0.5 M glycerol, the cell morphology changed to spore-like spherical cells, but the spores were not resistant to heat and sonication treatments. In contrast to the wild-type strain, the tatB–tatC mutant also showed a decreased cell growth rate and a lower maximum cell concentration. These results suggest possibility that the Tat system may contribute to export of various important proteins for development and growth for M. xanthus.     

Circulation of type 1 vaccine-derived poliovirus in the Philippines in 2001

In 2001, highly evolved type 1 circulating vaccine-derived poliovirus (cVDPV) was isolated from three acute flaccid paralysis patients and one contact from three separate communities in the Philippines. Complete genomic sequencing of these four cVDPV isolates revealed that the capsid region was derived from the Sabin 1 vaccine strain but most of the noncapsid region was derived from an unidentified enterovirus unrelated to the oral poliovirus vaccine (OPV) strains. The sequences of the cVDPV isolates were closely related to each other, and the isolates had a common recombination site. Most of the genetic and biological properties of the cVDPV isolates were indistinguishable from those of wild polioviruses. However, the most recently identified cVDPV isolate from a healthy contact retained the temperature sensitivity and partial attenuation phenotypes. The sequence relationships among the isolates and Sabin 1 suggested that cVDPV originated from an OPV dose given in 1998 to 1999 and that cVDPV circulated along a narrow chain of transmission. Type 1 cVDPV was last detected in the Philippines in September 2001, and population immunity to polio was raised by extensive OPV campaigns in late 2001 and early 2002.     

Proteomic Helicobacter pylori biomarkers discriminating between duodenal ulcer and gastric cancer

Protein patterns of 129 Helicobacter pylori strains isolated from Korean and Colombian patients suffering from duodenal ulcer or gastric cancer were analyzed by the high-throughput methodology SELDI-TOF-MS. Eighteen statistically significant candidate biomarkers discriminating between the two clinical outcomes were selected by using the Mann–Whitney test. Three biomarker proteins were purified and identified as a neutrophil-activating protein NapA (HU HPAG1_0821), a RNA-binding protein (HPAG1_0813), and a DNA-binding histone-like protein HU, respectively (jhp0228). These novel biomarkers can be used for development of diagnostic assays predicting the evolution to gastric cancer in H. pylori-infected patients.     

Mannose binding lectin and lung collectins interact with Toll-like receptor 4 and MD-2 by different mechanisms

Background

We have previously shown that lung collectins, surfactant protein A (SP-A) and surfactant protein D, interact with Toll-like receptor (TLR) 2, TLR4, or MD-2. Bindings of lung collectins to TLR2 and TLR4/MD-2 result in the alterations of signaling through these receptors, suggesting the immunomodulatory functions of lung collectins. Mannose binding lectin (MBL) is another collectin molecule which has structural homology to SP-A. The interaction between MBL and TLRs has not yet been determined.

Methods

We prepared recombinant MBL, and analyzed its bindings to recombinant soluble forms of TLR4 (sTLR4) and MD-2.

Results

MBL bound to sTLR4 and MD-2. The interactions were Ca²⁺-dependent and inhibited by mannose or monoclonal antibody against the carbohydrate-recognition domain of MBL. Treatment of sTLR4 or MD-2 by peptide N-glycosidase F significantly decreased the binding of MBL. SP-A bound to deglycosylated sTLR4, and this property did not change in chimeric molecules of SP-A/MBL in which Glu¹⁹⁵–Phe²²⁸ or Thr¹⁷⁴–Gly¹⁹⁴ of SP-A were replaced with the corresponding MBL sequences.

General Significance

These results suggested that MBL binds to TLR4 and MD-2 through the carbohydrate-recognition domain, and that oligosaccharide moieties of TLR4 and MD-2 are important for recognition by MBL. Since our previous studies indicated that lung collectins bind to the peptide portions of TLRs, MBL and lung collectins interact with TLRs by different mechanisms. These direct interactions between MBL and TLR4 or MD-2 suggest that MBL may modulate cellular responses by altering signals through TLRs.     

Structure and ligand binding properties of myoglobins reconstituted with monodepropionated heme: functional role of each heme propionate side chain

Two heme propionate side chains, which are attached at the 6 and 7 positions of the heme framework, are linked with Arg45 and Ser92, respectively, in sperm whale myoglobin. To evaluate the role of each propionate, two kinds of one-legged hemins, 6-depropionated and 7-depropionated protohemins, were prepared and inserted into the apomyoglobin to yield two reconstituted proteins. Structural data of the reconstituted myoglobins were obtained via an X-ray crystallographic analysis at a resolution of 1.1-1.4 A and resonance Raman spectroscopy. It was found that the lack of the 6-propionate reduces the number of hydrogen bonds in the distal site and clearly changes the position of the Arg45 residue with the disrupting Arg45-Asp60 interaction. In contrast, the removal of the 7-propionate does not cause a significant structural change in the residues of the distal and proximal sites. However, the resonance Raman studies suggested that the coordination bond strength of the His93-Fe bond for the protein with the 7-depropionated protoheme slightly increases compared to that for the protein with the native heme. The O2 and CO ligand binding studies for the reconstituted proteins with the one-legged hemes provide an important insight into the functional role of each propionate. The lack of the 6-propionate accelerates the O2 dissociation by ca. 3-fold compared to those of the other reconstituted and native proteins. The lack of the 7-propionate enhances the CO affinity by 2-fold compared to that of the protein with the native heme. These results indicate that the 6-propionate clearly contributes to the stabilization of the bound O2, whereas the 7-propionate plays an important role in the regulation of the Fe-His bond.     

Synthesis and conformational analysis of aib-containing peptide modelling for N-glycosylation site in N-glycoprotein

A tetrapetide containing an Aib residue, Boc-Asn-Aib-Thr-Aib-OMe, was synthesized as a peptide model for the N-glycosylation site in N-glycoproteins. Backbone conformation of the peptide and possible intramolecular interaction between the Asn and Thr side chains were elucidated by means of n.m.r. spectroscopy. Temperature dependence of NH proton chemical shift and NOE experiments showed that Boc-Asn-Aib-Thr-Aib-OMe has a tendency to form a β-turn structure with a hydrogen bond involving Thr and Aib⁴ NH groups. Incorporation of Aib residues in the peptide model promotes folding of the peptide backbone. With folded backbone conformation, carboxyamide protons of the Asn residue are not involved in hydrogen bond network, while the OH group of the Thr residue is a candidate for a hydrogen bond in DMSO-d₆ solution.     

Legumain/asparaginyl endopeptidase controls extracellular matrix remodeling through the degradation of fibronectin in mouse renal proximal tubular cells

Legumain/asparaginyl endopeptidase (EC 3.4.22.34) is a novel cysteine protease that is abundantly expressed in the late endosomes and lysosomes of renal proximal tubular cells. Recently, emerging evidence has indicated that legumain might play an important role in control of extracellular matrix turnover in various pathological conditions such as tumor growth/metastasis and progression of atherosclerosis. We initially found that purified legumain can directly degrade fibronectin, one of the main components of the extracellular matrix, in vitro. Therefore, we examined the effect of legumain on fibronectin degradation in cultured mouse renal proximal tubular cells. Fibronectin processing can be inhibited by chloroquine, an inhibitor of lysosomal degradation, and can be enhanced by the overexpression of legumain, indicating that fibronectin degradation occurs in the presence of legumain in lysosomes from renal proximal tubular cells. Furthermore, in legumain-deficient mice, unilateral ureteral obstruction (UUO)-induced renal interstitial protein accumulation of fibronectin and renal interstitial fibrosis were markedly enhanced. These findings indicate that legumain might have an important role in extracellular matrix remodeling via the degradation of fibronectin in renal proximal tubular cells.     

Drosophila beta 1,4-N-acetylgalactosaminyltransferase-A synthesizes the LacdiNAc structures on several glycoproteins and glycosphingolipids

The GalNAcbeta1,4GlcNAc (LacdiNAc or LDN) structure is a more common structural feature in invertebrate glycoconjugates when compared with the Galbeta1,4GlcNAc structure. Recently, beta1,4-N-acetylgalactosaminyltransferase (beta4GalNAcT) was identified in some invertebrates including Drosophila. However, the LDN structure has not been reported in Drosophila, and the biological function of LDN remains to be determined. In this study, we examined acceptor substrate specificity of Drosophila beta4GalNAcTA by using some N- and O-glycans on glycoproteins and neutral glycosphingolipids (GSLs). GalNAc was efficiently transferred toward N-glycans, O-glycans, and the arthro-series GSLs. Moreover, we showed that dbeta4GalNAcTA contributed to the synthesis of the LDN structure in vivo. The dbeta4GalNAcTA mRNA was highly expressed in the developmental and adult neuronal tissues. Thus, these results suggest that dbeta4GalNAcTA acts on the terminal GlcNAc residue of some glycans for the synthesis of LDN, and the LDN structure may play a role in the physiological or neuronal development of Drosophila.     

Functional and intracellular signaling differences associated with the Helicobacter pylori AlpAB adhesin from Western and East Asian strains

Following adhesion of Helicobacter pylori to gastric epithelial cells, intracellular signaling leads to cytokine production, which causes H. pylori-related gastric injury. Two adjacent homologous genes (alpA and alpB), which encode H. pylori outer membrane proteins, are thought to be associated with adhesion and cytokine induction. We co-cultured gastric epithelial cells with wild type H. pylori strains and their corresponding alpA/alpB-deleted mutants (DeltaalpAB). Results were confirmed by complementation. Flow cytometry confirmed that AlpAB was involved in cellular adhesion. Deletion of alpAB reduced interleukin (IL)-6 induction in gastric epithelial cells. Deletion of alpAB reduced IL-8 induction with East Asian but not with Western strains. All AlpAB-positive strains tested activated the extracellular signal-regulated kinase, c-Fos, and cAMP-responsive element-binding protein. Activation of the Jun-N-terminal kinase, c-Jun, and NF-kappaB was exclusive to AlpAB from East Asian strains. DeltaalpAB mutants poorly colonized the stomachs of C57BL/6 mice and were associated with lower mucosal levels of KC and IL-6. Our results suggest that AlpAB may induce gastric injury by mediating adherence to gastric epithelial cells and by modulating proinflammatory intracellular signaling cascades. Known geographical differences in H. pylori-related clinical outcomes may relate to differential effects of East Asian and Western types of AlpAB on NF-kappaB-related proinflammatory signaling pathways.