UDP-Gal: GlcNAc-R β1,4-galactosyltransferase—a target enzyme for drug design. Acceptor specificity and inhibition of the enzyme

Galactosyltransferases are important enzymes for the extension of the glycan chains of glycoproteins and glycolipids, and play critical roles in cell surface functions and in the immune system. In this work, the acceptor specificity and several inhibitors of bovine β1,4-Gal-transferase T1 (β4GalT, EC 2.4.1.90) were studied. Series of analogs of N-acetylglucosamine (GlcNAc) and GlcNAc-carrying glycopeptides were synthesized as acceptor substrates. Modifications were made at the 3-, 4- and 6-positions of the sugar ring of the acceptor, in the nature of the glycosidic linkage, in the aglycone moiety and in the 2-acetamido group. The acceptor specificity studies showed that the 4-hydroxyl group of the sugar ring was essential for β4GalT activity, but that the 3-hydroxyl could be replaced by an electronegative group. Compounds having the anomeric β-configuration were more active than those having the α-configuration, and O-, S- and C-glycosyl compounds were all active as substrates. The aglycone was a major determinant for the rate of Gal-transfer. Derivatives containing a 2-naphthyl aglycone were inactive as substrates although quinolinyl groups supported activity. Several compounds having a bicyclic structure as the aglycone were found to bind to the enzyme and inhibited the transfer of Gal to control substrates. The best small hydrophobic GlcNAc-analog inhibitor was found to be 1-thio-N-butyrylGlcNβ-(2-naphthyl) with a K_i of 0.01 mM. These studies help to delineate β4GalT–substrate interactions and will aid in the development of biologically applicable inhibitors of the enzyme.     

Comparative genomic evidence for a close relationship between the dimorphic prosthecate bacteria Hyphomonas neptunium and Caulobacter crescentus

The dimorphic prosthecate bacteria (DPB) are alpha-proteobacteria that reproduce in an asymmetric manner rather than by binary fission and are of interest as simple models of development. Prior to this work, the only member of this group for which genome sequence was available was the model freshwater organism Caulobacter crescentus. Here we describe the genome sequence of Hyphomonas neptunium, a marine member of the DPB that differs from C. crescentus in that H. neptunium uses its stalk as a reproductive structure. Genome analysis indicates that this organism shares more genes with C. crescentus than it does with Silicibacter pomeroyi (a closer relative according to 16S rRNA phylogeny), that it relies upon a heterotrophic strategy utilizing a wide range of substrates, that its cell cycle is likely to be regulated in a similar manner to that of C. crescentus, and that the outer membrane complements of H. neptunium and C. crescentus are remarkably similar. H. neptunium swarmer cells are highly motile via a single polar flagellum. With the exception of cheY and cheR, genes required for chemotaxis were absent in the H. neptunium genome. Consistent with this observation, H. neptunium swarmer cells did not respond to any chemotactic stimuli that were tested, which suggests that H. neptunium motility is a random dispersal mechanism for swarmer cells rather than a stimulus-controlled navigation system for locating specific environments. In addition to providing insights into bacterial development, the H. neptunium genome will provide an important resource for the study of other interesting biological processes including chromosome segregation, polar growth, and cell aging.     

Site directed processing: Role of amino acid sequences and glycosylation of acceptor glycopeptides in the assembly of extended mucin type O-glycan core 2

Background

The assembly of Ser/Thr-linked O-glycans of mucins with core 2 structures is initiated by polypeptide GalNAc-transferase (ppGalNAc-T), followed by the action of core 1 β3-Gal-transferase (C1GalT) and core 2 β6-GlcNAc-transferase (C2GnT). β4-Gal-transferase (β4GalT) extends core 2 and forms the backbone structure for biologically important epitopes. O-glycan structures are often abnormal in chronic diseases. The goal of this work is to determine if the activity and specificity of these enzymes are directed by the sequences and glycosylation of substrates.

Methods

We studied the specificities of four enzymes that synthesize extended O-glycan core 2 using as acceptor substrates synthetic mucin derived peptides and glycopeptides, substituted with GalNAc or O-glycan core structures 1, 2, 3, 4 and 6.

Results

Specific Thr residues were found to be preferred sites for the addition of GalNAc, and Pro in the + 3 position was found to especially enhance primary glycosylation. An inverse relationship was found between the size of adjacent glycans and the rate of GalNAc addition. All four enzymes could distinguish between substrates having different amino acid sequences and O-glycosylated sites. A short glycopeptide Galβ1–3GalNAcα-TAGV was identified as an efficient C2GnT substrate.

Conclusions

The activities of four enzymes assembling the extended core 2 structure are affected by the amino acid sequence and presence of carbohydrates on nearby residues in acceptor glycopeptides. In particular, the sequences and O-glycosylation patterns direct the addition of the first and second sugar residues by ppGalNAc-T and C1GalT which act in a site directed fashion.

General significance

Knowledge of site directed processing enhances our understanding of the control of O-glycosylation in normal cells and in disease.     

Survival of Campylobacter jejuni under Conditions of Atmospheric Oxygen Tension with the Support of Pseudomonas spp.

Campylobacter jejuni is a major food-borne pathogen. Despite causing enteritis in humans, it is a well-adapted intestinal microorganism in animals, hardly ever generating disease symptoms. Nevertheless, as a true microaerophilic microorganism it is still puzzling how Campylobacter cells can survive on chicken meat, the main source of human infection. In this study, we demonstrate that C. jejuni is able to withstand conditions of atmospheric oxygen tension when cocultured with Pseudomonas species, major food-spoiling bacteria that are frequently found on chicken meat in rather high numbers. Using an in vitro survival assay, interactions of 145 C. jejuni wild-type strains and field isolates from chicken meat, broiler feces, and human clinical samples with type strains and food isolates of Pseudomonas spp., Proteus mirabilis, Citrobacter freundii, Micrococcus luteus, and Enterococcus faecalis were studied. When inoculated alone or in coculture with Proteus mirabilis, Citrobacter freundii, Micrococcus luteus, or Enterococcus faecalis type strains, Campylobacter cells were able to survive ambient oxygen levels for no more than 18 h. In contrast, Campylobacter bacteria inoculated with type strains or wild-type isolates of Pseudomonas showed a prolonged aerobic survival of up to >48 h. This microbial commensalism was diverse in C. jejuni isolates from different sources; isolates from chicken meat and humans in coculture with Pseudomonas putida were able to use this survival support better than fecal isolates from broilers. Scanning electron microscopy revealed the development of fiberlike structures braiding P. putida and C. jejuni cells. Hence, it seems that microaerophilic C. jejuni is able to survive ambient atmospheric oxygen tension by metabolic commensalism with Pseudomonas spp. This bacterium-bacterium interaction might set the basis for survival of C. jejuni on chicken meat and thus be the prerequisite step in the pathway toward human infection.Campylobacter food-borne infections are the most prevalent bacterial enteric infections in humans in industrialized and developing countries (1). It has been shown that most human infections are related to poultry meat and food produced from cattle or sheep (34, 41). Campylobacter jejuni, the species most frequently causing human disease, can be isolated from the animal intestinal tract at levels of up to 10⁹ CFU per gram of feces and can thus be called a well-adapted intestinal microorganism (30, 37). Nevertheless, because it causes human disease as a food-borne pathogen, it has to survive outside the gut. By cross-contamination at the level of the abattoir, Campylobacter bacteria hit the meat surface and have to adapt to different environmental challenges. C. jejuni is a true microaerophilic bacterium; thus, on the one hand it requires oxygen, but on the other hand it cannot grow under normal atmospheric oxygen tension conditions (15). Despite its sensitivity to high oxygen tension in vitro, viable and culturable Campylobacter bacteria can be isolated from nonskinned chicken meat at frequencies of 10⁴ CFU/g (9, 19). Assumptions on the mechanisms by which Campylobacter cells survive on meat surfaces are diverse, for example, by growing in biofilms, entering a “viable but nonculturable state,” or interacting with other microorganisms.For instance, C. jejuni is able to resist protozoa digestion and can parasitize inside protozoa, e.g., Tetrahymena pyriformis (35). This mechanism provides survival in harsh environments and resistance to antimicrobial substances and thus enhances the potential for transmission. But bacterium-bacterium interaction has also been demonstrated to be of a high level of importance for intestinal survival and uptake (20). Accordingly, members of Campylobacter have been identified to initiate cellular uptake of commensal bacteria into enterocytes (14). However, a bacterial community can also mean competition, e.g., bacteriocin production by Lactobacillus salivarius that is effective against Campylobacter colonization (36).Meat surfaces harbor numerous bacterial species (24). Some of these bacteria have adapted to this specific environmental niche and are well-known spoilage bacteria. Most relevant species belong to the family Pseudomonadaceae. But also different members of the Enterobacteriaceae can be found on meat. To date, information regarding the interaction between spoilage bacteria and pathogens is of increasing importance for public health safety measures.Hence, experimental data on the survival of C. jejuni isolates in the presence of selected meat-spoiling bacteria were analyzed and clearly demonstrated a specific interaction with type strains and isolates of Pseudomonas putida, Pseudomonas fragi, and Pseudomonas fluorescens from chicken meat surfaces.     

Powerful workhorses for antimicrobial peptide expression and characterization

Discovery of antimicrobial peptides (AMP) is to a large extent based on screening of fractions of natural samples in bacterial growth inhibition assays. However, the use of bacteria is not limited to screening for antimicrobial substances. In later steps, bioengineered "bugs" can be applied to both production and characterization of AMPs. Here we describe the idea to use genetically modified Escherichia coli strains for both these purposes. This approach allowed us to investigate SpStrongylocins 1 and 2 from the purple sea urchin Strongylocentrotus purpuratus only based on sequence information from a cDNA library and without previous direct isolation or chemical synthesis of these peptides. The recombinant peptides are proved active against all bacterial strains tested. An assay based on a recombinant E. coli sensor strain expressing insect luciferase, revealed that SpStrongylocins are not interfering with membrane integrity and are therefore likely to have intracellular targets.     

Potentials of mean force for protein structure prediction vindicated, formalized and generalized

Understanding protein structure is of crucial importance in science, medicine and biotechnology. For about two decades, knowledge-based potentials based on pairwise distances--so-called "potentials of mean force" (PMFs)--have been center stage in the prediction and design of protein structure and the simulation of protein folding. However, the validity, scope and limitations of these potentials are still vigorously debated and disputed, and the optimal choice of the reference state--a necessary component of these potentials--is an unsolved problem. PMFs are loosely justified by analogy to the reversible work theorem in statistical physics, or by a statistical argument based on a likelihood function. Both justifications are insightful but leave many questions unanswered. Here, we show for the first time that PMFs can be seen as approximations to quantities that do have a rigorous probabilistic justification: they naturally arise when probability distributions over different features of proteins need to be combined. We call these quantities "reference ratio distributions" deriving from the application of the "reference ratio method." This new view is not only of theoretical relevance but leads to many insights that are of direct practical use: the reference state is uniquely defined and does not require external physical insights; the approach can be generalized beyond pairwise distances to arbitrary features of protein structure; and it becomes clear for which purposes the use of these quantities is justified. We illustrate these insights with two applications, involving the radius of gyration and hydrogen bonding. In the latter case, we also show how the reference ratio method can be iteratively applied to sculpt an energy funnel. Our results considerably increase the understanding and scope of energy functions derived from known biomolecular structures.     

Global responses of Aliivibrio salmonicida to hydrogen peroxide as revealed by microarray analysis

Aliivibrio salmonicida causes "cold-water vibriosis" (or "Hitra disease") in fish, including marine-reared Atlantic salmon. During development of the disease the bacterium will encounter macrophages with antibacterial activities such as production of damaging reactive oxygen species (ROS). To defend itself the bacterium will presumably start producing detoxifying enzymes, reducing agents, and proteins involved in DNA and protein repair systems. Even though responses to oxidative stress are well studied for a few model bacteria, little work has been done in general to explain how important groups of pathogens, like members of the Vibrionaceae family, can survive at high levels of ROS. We have used bioinformatic tools and microarray to study how A. salmonicida responds to hydrogen peroxide (H(2)O(2)). First, we used the recently published genome sequence to predict potential binding sites for OxyR (H(2)O(2) response regulator). The computer-based search identified OxyR sites associated with 20 single genes and 8 operons, and these predictions were compared to experimental data from Northern blot analysis and microarray analysis. In general, OxyR binding site predictions and experimental results are in agreement. Up- and down regulated genes are distributed among all functional gene categories, but a striking number of ≥2 fold up regulated genes encode proteins involved in detoxification and DNA repair, are part of reduction systems, or are involved in carbon metabolism and regeneration of NADPH. Our predictions and -omics data corroborates well with findings from other model bacteria, but also suggest species-specific gene regulation.     

Structures and structure-activity relationships of three mitogenic and complement fixing pectic arabinogalactans from the malian antiulcer plants Cochlospermum tinctorium A. Rich and Vernonia kotschyana Sch. Bip. ex Walp

Structures of three pectic arabinogalactans, one from Vernonia kotschyana (Vk2a) and two from Cochlospermum tinctorium (Ct50A1 and Ct50A2), and their complement fixation and induction of B cell proliferation in vitro were compared. The polysaccharide Vk2a expressed potent biological activity in both assays compared with Ct50A1 and Ct50A2. Vk2a possessed a very high molecular weight (1150 +/- 20 kDa) compared with Ct50A1 and Ct50A2 which both showed a polydisperse nature with the highest molecular weight polymers in each fraction estimated at approximately 105 kDa (Ct1a) and 640 +/- 100 kDa (Ct2a), respectively. The HMW polymers showed complement fixation in the same range as the native fractions. The arabinogalactan II content was low in Vk2a (2%) compared with that in Ct50A1 (23%) and Ct50A2 (12%). The high molecular weight polymers were subjected to digestion with a beta-d-(1, 3)-galactanase-rich fraction from Driselase, oligomers were isolated by HPAEC, and their finer structures were determined by MALDI- and ES-qoToF-MS, linkage, and monosaccharide composition analyses. Vk2a consists of both a galacturonan core and a rhamnogalacturonan core rich in neutral side chains. The backbones of both Ct-polysaccharides consist mainly of RG-I regions with numerous neutral side chains dominated by galactosyl residues, whereas the homogalacturonan regions seem to be small. Differences in the chain lengths of the 6-linked galacto-oligosaccharides attached to the 3-linked galactan core could not be related to the differences in the potencies of the biological activities observed.