Localization and structural analysis of a conserved pyruvylated epitope in Bacillus anthracis secondary cell wall polysaccharides and characterization of the galactose-deficient wall polysaccharide from avirulent B. anthracis CDC 684

Bacillus anthracis CDC 684 is a naturally occurring, avirulent variant and close relative of the highly pathogenic B. anthracis Vollum. Bacillus anthracis CDC 684 contains both virulence plasmids, pXO1 and pXO2, yet is non-pathogenic in animal models, prompting closer scrutiny of the molecular basis of attenuation. We structurally characterized the secondary cell wall polysaccharide (SCWP) of B. anthracis CDC 684 (Ba684) using chemical and NMR spectroscopy analysis. The SCWP consists of a HexNAc trisaccharide backbone having identical structure as that of B. anthracis Pasteur, Sterne and Ames, →4)-β-d-ManpNAc-(1?→?4)-β-d-GlcpNAc-(1?→?6)-α-d-GlcpNAc-(1→. Remarkably, although the backbone is fully polymerized, the SCWP is the devoid of all galactosyl side residues, a feature which normally comprises 50% of the glycosyl residues on the highly galactosylated SCWPs from pathogenic strains. This observation highlights the role of defective wall assembly in virulence and indicates that polymerization occurs independently of galactose side residue attachment. Of particular interest, the polymerized Ba684 backbone retains the substoichiometric pyruvate acetal, O-acetate and amino group modifications found on SCWPs from normal B. anthracis strains, and immunofluorescence analysis confirms that SCWP expression coincides with the ability to bind the surface layer homology (SLH) domain containing S-layer protein extractable antigen-1. Pyruvate was previously demonstrated as part of a conserved epitope, mediating SLH-domain protein attachment to the underlying peptidoglycan layer. We find that a single repeating unit, located at the distal (non-reducing) end of the Ba684 SCWP, is structurally modified and that this modification is present in identical manner in the SCWPs of normal B. anthracis strains. These polysaccharides terminate in the sequence: (S)-4,6-O-(1-carboxyethylidene)-β-d-ManpNAc-(1?→?4)-[3-O-acetyl]-β-d-GlcpNAc-(1?→?6)-α-d-GlcpNH(2)-(1→.     

Crystal structure of human cystatin D, a cysteine peptidase inhibitor with restricted inhibition profile

Cystatins are natural inhibitors of papain-like (family C1) and legumain-related (family C13) cysteine peptidases. Cystatin D is a type 2 cystatin, a secreted inhibitor found in human saliva and tear fluid. Compared with its homologues, cystatin D presents an unusual inhibition profile with a preferential inhibition cathepsin S > cathepsin H > cathepsin L and no inhibition of cathepsin B or pig legumain. To elucidate the structural reasons for this specificity, we have crystallized recombinant human Arg(26)-cystatin D and solved its structures at room temperature and at cryo conditions to 2.5- and 1.8-A resolution, respectively. Human cystatin D presents the typical cystatin fold, with a five-stranded anti-parallel beta-sheet wrapped around a five-turn alpha-helix. The structures reveal differences in the peptidase-interacting regions when compared with other cystatins, providing plausible explanations for the restricted inhibitory specificity of cystatin D for some papain-like peptidases and its lack of reactivity toward legumain-related enzymes.     

The effects of methyltestosteone on reproductive function in male greyhounds

Oral administration of methyltestosterone (MT) at 50 mg/dog/day to intact adult male greyhounds for 90 d resulted in decreased (P < 0.05) mean daily sperm output and mean testicular length. Additionally, the mean diameter of seminiferous tubules in MT-treated dogs tended to decrease (P = 0.08). Mean concentrations of luteinizing hormone (LH) and follicle stimulating hormone (FSH) and concentrations of testosterone in serum were also decreased or tended to decrease (P = 0.0003 to 0.059) at various sampling periods during MT treatment, suggesting alterations in spermatogenesis resulted from decreased serum concentrations of gonadotropins and steroids. Mean daily sperm output, mean testicular length, mean seminiferous tubule diameter and mean concentrations of FSH in serum were not decreased (P > 0.05) at the end of a 90-d recovery period. However, mean concentrations of serum LH and concentrations of testosterone were still lower (P < 0.05) during five of six and one of six sampling times, respectively, during the recovery period than the pretreatment levels, suggesting a prolonged effect of MT treatment on the pituitary/gonadal axis.     

Self‐rescue of an EXTENSIN mutant reveals alternative gene expression programs and candidate proteins for new cell wall assembly in Arabidopsis

Plants encode a poorly understood superfamily of developmentally expressed cell wall hydroxyproline‐rich glycoproteins (HRGPs). One, EXTENSIN3 (EXT3) of the 168 putative HRGPs, is critical in the first steps of new wall assembly, demonstrated by broken and misplaced walls in its lethal homozygous mutant. Here we report the findings of phenotypic (not genotypic) revertants of the ext3 mutant and in‐depth analysis including microarray and qRT‐PCR (polymerase chain reaction). The aim was to identify EXT3 substitute(s), thus gaining a deeper understanding of new wall assembly. The data show differential expression in the ext3 mutant that included 61% (P ≤ 0.05) of the HRGP genes, and ability to self‐rescue by reprogramming expression. Independent revertants had reproducible expression networks, largely heritable over the four generations tested, with some genes displaying transgenerational drift towards wild‐type expression levels. Genes for nine candidate regulatory proteins as well as eight candidate HRGP building materials and/or facilitators of new wall assembly or maintenance, in the (near) absence of EXT3 expression, were identified. Seven of the HRGP fit the current model of EXT function. In conclusion, the data on phenotype comparisons and on differential expression of the genes‐of‐focus provide strong evidence that different combinations of HRGPs regulated by alternative gene expression networks, can make functioning cell walls, resulting in (apparently) normal plant growth and development. More broadly, this has implications for interpreting the cause of any mutant phenotype, assigning gene function, and genetically modifying plants for utilitarian purposes.     

Intestinal Origin of Sourdough Lactobacillus reuteri Isolates as Revealed by Phylogenetic, Genetic, and Physiological Analysis

Lactobacillus reuteri is both a gut symbiont and a stable member of sourdough microbiota. This study employed multilocus sequence analysis and an analysis of host-specific physiological and genetic traits to assign five sourdough isolates to rodent- or human-specific lineages. Comparative genome hybridization revealed that the model sourdough isolate LTH2584 had a genome content very similar to that of the model rodent isolate 100-23. These results demonstrate that sourdough isolates of L. reuteri are of intestinal origin.     

The effects of the C-terminal amidation of mastoparans on their biological actions and interactions with membrane-mimetic systems

Polycationic peptides may present their C-termini in either amidated or acidic form; however, the effects of these conformations on the mechanisms of interaction with the membranes in general were not properly investigated up to now. Protonectarina-MP mastoparan with an either amidated or acidic C-terminus was utilized to study their interactions with anionic and zwitterionic vesicles, using measurements of dye leakage and a combination of H/D exchange and mass spectrometry to monitor peptide–membrane interactions. Mast cell degranulation, hemolysis and antibiosis assays were also performed using these peptides, and the results were correlated with the structural properties of the peptides. The C-terminal amidation promotes the stabilization of the secondary structure of the peptide, with a relatively high content of helical conformations, permitting a deeper interaction with the phospholipid constituents of animal and bacterial cell membranes. The results suggested that at low concentrations Protonectarina-MP interacts with the membranes in a way that both terminal regions remain positioned outside the external surface of the membrane, while the α-carbon backbone becomes partially embedded in the membrane core and changing constantly the conformation, and causing membrane destabilization. The amidation of the C-terminal residue appears to be responsible for the stabilization of the peptide conformation in a secondary structure that is richer in α-helix content than its acidic congener. The helical, amphipathic conformation, in turn, allows a deeper peptide–membrane interaction, favoring both biological activities that depend on peptide structure recognition by the GPCRs (such as exocytosis) and those activities dependent on membrane perturbation (such as hemolysis and antibiosis).     

Co-distribution of annexin VI and actin in secretory ameloblasts and odontoblasts of rat incisor

Summary Annexin VI and actin were detected by immunoblot analysis in the enamel- and dentin-related portions of dental tissues. Annexin VI was found mainly in the particulate fraction whereas actin was detected in both the soluble and particulate fractions. By immunoelectron microscopy, annexin VI antibodies conjugated with colloidal gold were seen to label the mitochondria, the cytosol and the nucleus of secretory ameloblasts and odontoblasts of rat incisor. In the processes of these cell, the plasmalemmal undercoat was labeled. Antiactin antibodies labeled the desmosome-like junctions, the cytosol, and the mitochondria of the cell bodies. Extensive labeling was seen at the periphery of the Tomes' processes and odontoblast processes. These results suggest that annexin VI may play a role in Ca²⁺-regulation in the cell bodies, especially as a calcium receptor protein in the mitochondria. Moreover, annexin VI and actin seem to be co-distributed in secretory processes. Thus, these proteins might be both involved in exocytotic and endocytotic events.     

Isolation and characterization of deteriosomes from rat liver

Deteriosomes, a new class of microvesicles, have been isolated from rat liver tissue. These microvesicles are similar to those isolated previously from plant tissue [Yao et al., Proc Natl Acad Sci USA 88:2269–2273, 1991] in that they are nonsedimentable and enriched in membrane catabolites, particularly products of phospholipid degradation. Liver deteriosomes range in size from 0.05 μm to 0.11 μm in radius. They are also much more permeable than microsomal membrane vesicles indicating that the deteriosome bilayer is perturbed. The data are consistent with the proposal that deteriosomes are formed from membranes by microvesiculation and that they represent an intermediate stage of membrane deterioration. Furthermore, liver deteriosomes were found to contain phospholipase A₂ activity. This suggests that they not only serve as a means of moving destabilizing macromolecular catabolites out of membranes into the cytosol but also possess enzymatic activity. The fact that the specific activity of phospholipase A₂ is higher in deteriosomes than in deteriosome-free cytosol suggests that some of the enzymatic activity traditionally assumed to be cytosolic may in fact be associated with deteriosomes.     

Molecular Basis of Calpain Cleavage and Inactivation of the Sodium-Calcium Exchanger 1 in Heart Failure

Cardiac sodium (Na⁺)-calcium (Ca²⁺) exchanger 1 (NCX1) is central to the maintenance of normal Ca²⁺ homeostasis and contraction. Studies indicate that the Ca²⁺-activated protease calpain cleaves NCX1. We hypothesized that calpain is an important regulator of NCX1 in response to pressure overload and aimed to identify molecular mechanisms and functional consequences of calpain binding and cleavage of NCX1 in the heart. NCX1 full-length protein and a 75-kDa NCX1 fragment along with calpain were up-regulated in aortic stenosis patients and rats with heart failure. Patients with coronary artery disease and sham-operated rats were used as controls. Calpain co-localized, co-fractionated, and co-immunoprecipitated with NCX1 in rat cardiomyocytes and left ventricle lysate. Immunoprecipitations, pull-down experiments, and extensive use of peptide arrays indicated that calpain domain III anchored to the first Ca²⁺ binding domain in NCX1, whereas the calpain catalytic region bound to the catenin-like domain in NCX1. The use of bioinformatics, mutational analyses, a substrate competitor peptide, and a specific NCX1-Met³⁶⁹ antibody identified a novel calpain cleavage site at Met³⁶⁹. Engineering NCX1-Met³⁶⁹ into a tobacco etch virus protease cleavage site revealed that specific cleavage at Met³⁶⁹ inhibited NCX1 activity (both forward and reverse mode). Finally, a short peptide fragment containing the NCX1-Met³⁶⁹ cleavage site was modeled into the narrow active cleft of human calpain. Inhibition of NCX1 activity, such as we have observed here following calpain-induced NCX1 cleavage, might be beneficial in pathophysiological conditions where increased NCX1 activity contributes to cardiac dysfunction.     

The O-Hyp glycosylation code in tobacco and Arabidopsis and a proposed role of Hyp-glycans in secretion

Most aspects of plant growth involve cell surface hydroxyproline (Hyp)-rich glycoproteins (HRGPs) whose properties depend on arabinogalactan polysaccharides and arabinosides that define the molecular surface. Potential glycosylation sites are defined by an O-Hyp glycosylation code: contiguous Hyp directs arabinosylation. Clustered non-contiguous Hyp directs arabinogalactosylation. Elucidation of this code involved a single species, tobacco (Nicotiana tabacum) BY-2 cells. However, recent work suggests species variation, perhaps tissue specific Hyp glycosylation. Thus, the extent to which the Hyp glycosylation code is 'global' needs testing. We compared the ability of distantly related Arabidopsis cell cultures to process putative HRGP glycosylation motifs encoded by synthetic genes. The genes included: repetitive Ser-Pro, Ser-Pro2, Ser-Pro4 and an analog of the tomato arabinogalactan-protein, LeAGP-1DeltaGPI. All were expressed as enhanced green fluorescent protein (EGFP) fusion glycoproteins, designated: AtSO-EGFP (O=Hyp), AtSO2-EGFP, AtSO4-EGFP and AtEGFP-LeAGP-1DeltaGPI, respectively. The Arabidopsis glycosylation patterns were essentially similar to those observed in Nicotiana: non-contiguous Hyp residues in AtSO-EGFP were glycosylated exclusively with arabinogalactan polysaccharides while contiguous Hyp in AtSO2-EGFP and AtSO4-EGFP was exclusively arabinosylated. Mixed contiguous and non-contiguous Hyp residues in AtEGFP-LeAGP-1DeltaGPI were also arabinosylated and arabinogalactosylated consistent with the code. However, slightly more arabinogalactosylated Hyp and less non-glycosylated Hyp in AtEGFP-LeAGP-1DeltaGPI than tobacco NtEGFP-LeAGP-1DeltaGPI suggested Arabidopsis prolyl hydroxylases have a slightly broader specificity. Arabidopsis Hyp-arabinogalactans differed from tobacco in decreased glucuronic acid content and lack of rhamnose. Yields of the EGFP fusion glycoproteins were dramatically higher than targeted EGFP lacking Hyp-glycomodules. This validates earlier suggestions that the glycosylation of proteins facilitates their secretion.