N-glycans of the porcine nematode parasite Ascaris suum are modified with phosphorylcholine and core fucose residues

In recent years, the glycoconjugates of many parasitic nematodes have attracted interest due to their immunogenic and immunomodulatory nature. Previous studies with the porcine roundworm parasite Ascaris suum have focused on its glycosphingolipids, which were found, in part, to be modified by phosphorylcholine. Using mass spectrometry and western blotting, we have now analyzed the peptide N-glycosidase A-released N-glycans of adults of this species. The presence of hybrid bi- and triantennary N-glycans, some modified by core alpha1,6-fucose and peripheral phosphorylcholine, was demonstrated by LC/electrospray ionization (ESI)-Q-TOF-MS/MS, as was the presence of paucimannosidic N-glycans, some of which carry core alpha1,3-fucose, and oligomannosidic oligosaccharides. Western blotting verified the presence of protein-bound phosphorylcholine and core alpha1,3-fucose, whereas glycosyltransferase assays showed the presence of core alpha1,6-fucosyltransferase and Lewis-type alpha1,3-fucosyltransferase activities. Although, the unusual tri- and tetrafucosylated glycans found in the model nematode Caenorhabditis elegans were not found, the vast majority of the N-glycans found in A. suum represent a subset of those found in C. elegans; thus, our data demonstrate that the latter is an interesting glycobiological model for parasitic nematodes.     

Dynamics driving function: new insights from electron transferring flavoproteins and partner complexes

Electron transferring flavoproteins (ETFs) are soluble heterodimeric FAD-containing proteins that function primarily as soluble electron carriers between various flavoprotein dehydrogenases. ETF is positioned at a key metabolic branch point, responsible for transferring electrons from up to 10 primary dehydrogenases to the membrane-bound respiratory chain. Clinical mutations of ETF result in the often fatal disease glutaric aciduria type II. Structural and biophysical studies of ETF in complex with partner proteins have shown that ETF partitions the functions of partner binding and electron transfer between (a) a 'recognition loop', which acts as a static anchor at the ETF-partner interface, and (b) a highly mobile redox-active FAD domain. Together, this enables the FAD domain of ETF to sample a range of conformations, some compatible with fast interprotein electron transfer. This 'conformational sampling' enables ETF to recognize structurally distinct partners, whilst also maintaining a degree of specificity. Complex formation triggers mobility of the FAD domain, an 'induced disorder' mechanism contrasting with the more generally accepted models of protein-protein interaction by induced fit mechanisms. We discuss the implications of the highly dynamic nature of ETFs in biological interprotein electron transfer. ETF complexes point to mechanisms of electron transfer in which 'dynamics drive function', a feature that is probably widespread in biology given the modular assembly and flexible nature of biological electron transfer systems.     

Analysis of the NADH-dependent retinaldehyde reductase activity of amphioxus retinol dehydrogenase enzymes enhances our understanding of the evolution of the retinol dehydrogenase family

In vertebrates, multiple microsomal retinol dehydrogenases are involved in reversible retinol/retinal interconversion, thereby controlling retinoid metabolism and retinoic acid availability. The physiologic functions of these enzymes are not, however, fully understood, as each vertebrate form has several, usually overlapping, biochemical roles. Within this context, amphioxus, a group of chordates that are simpler, at both the functional and genomic levels, than vertebrates, provides a suitable evolutionary model for comparative studies of retinol dehydrogenase enzymes. In a previous study, we identified two amphioxus enzymes, Branchiostoma floridae retinol dehydrogenase 1 and retinol dehydrogenase 2, both candidates to be the cephalochordate orthologs of the vertebrate retinol dehydrogenase enzymes. We have now proceeded to characterize these amphioxus enzymes. Kinetic studies have revealed that retinol dehydrogenase 1 and retinol dehydrogenase 2 are microsomal proteins that catalyze the reduction of all-trans-retinaldehyde using NADH as cofactor, a remarkable combination of substrate and cofactor preferences. Moreover, evolutionary analysis, including the amphioxus sequences, indicates that Rdh genes were extensively duplicated after cephalochordate divergence, leading to the gene cluster organization found in several mammalian species. Overall, our data provide an evolutionary reference with which to better understand the origin, activity and evolution of retinol dehydrogenase enzymes.     

Effects of different levels of ergot in concentrates on the growing and slaughtering performance of bulls and on carry-over into edible tissue

The aim of the present study was to examine long-term effects of low levels of ergot alkaloids on growing bulls. Natural grown ergot with a mean total alkaloid concentrations of 633 mg/kg, and ergotamine (25%), ergocristine (15%) and ergosine (13%) as the most prominent alkaloids, was used. In a dose-response study 38 Holstein Friesian bulls were fed with three different doses of this ergot (0, 0.45 and 2.25 g/kg concentrate corresponding to an average total alkaloid concentration of the daily ration of 0, 69 and 421 microg/kg DM) over a period of approximately 230 days. Live weight, feed intake and health condition were monitored over the entire test period. The bulls were slaughtered at a live weight of approximately 550 kg. Carcass composition and quality were recorded and samples of liver, muscle, kidneys, fat, bile, urine and blood were analysed for ergot alkaloids. Liver enzyme activities and total bilirubin were measured in the blood. Statistically, no significant differences were detectable between the three feeding groups. Mean live weight gain over all groups was 1.41 kg/d with a mean dry matter intake of 7.35 kg/d. No carry over into tissues could be proved out of the experiment. To derive a no-effect level for beef cattle further research including higher ergot doses will be necessary.     

Mapping of the active site of glutamate carboxypeptidase II by site-directed mutagenesis

Human glutamate carboxypeptidase II [GCPII (EC 3.4.17.21)] is recognized as a promising pharmacological target for the treatment and imaging of various pathologies, including neurological disorders and prostate cancer. Recently reported crystal structures of GCPII provide structural insight into the organization of the substrate binding cavity and highlight residues implicated in substrate/inhibitor binding in the S1' site of the enzyme. To complement and extend the structural studies, we constructed a model of GCPII in complex with its substrate, N-acetyl-l-aspartyl-l-glutamate, which enabled us to predict additional amino acid residues interacting with the bound substrate, and used site-directed mutagenesis to assess the contribution of individual residues for substrate/inhibitor binding and enzymatic activity of GCPII. We prepared and characterized 12 GCPII mutants targeting the amino acids in the vicinity of substrate/inhibitor binding pockets. The experimental results, together with the molecular modeling, suggest that the amino acid residues delineating the S1' pocket of the enzyme (namely Arg210) contribute primarily to the high affinity binding of GCPII substrates/inhibitors, whereas the residues forming the S1 pocket might be more important for the 'fine-tuning' of GCPII substrate specificity.     

Properties of pyranose dehydrogenase purified from the litter-degrading fungus Agaricus xanthoderma

We purified an extracellular pyranose dehydrogenase (PDH) from the basidiomycete fungus Agaricus xanthoderma using ammonium sulfate fractionation and ion-exchange and hydrophobic interaction chromatography. The native enzyme is a monomeric glycoprotein (5% carbohydrate) containing a covalently bound FAD as its prosthetic group. The PDH polypeptide consists of 575 amino acids and has a molecular mass of 65 400 Da as determined by MALDI MS. On the basis of the primary structure of the mature protein, PDH is a member of the glucose-methanol-choline oxidoreductase family. We constructed a homology model of PDH using the 3D structure of glucose oxidase from Aspergillus niger as a template. This model suggests a novel type of bi-covalent flavinylation in PDH, 9-S-cysteinyl, 8-alpha-N3-histidyl FAD. The enzyme exhibits a broad sugar substrate tolerance, oxidizing structurally different aldopyranoses including monosaccharides and oligosaccharides as well as glycosides. Its preferred electron donor substrates are D-glucose, D-galactose, L-arabinose, and D-xylose. As shown by in situ NMR analysis, D-glucose and D-galactose are both oxidized at positions C2 and C3, yielding the corresponding didehydroaldoses (diketoaldoses) as the final reaction products. PDH shows no detectable activity with oxygen, and its reactivity towards electron acceptors is rather limited, reducing various substituted benzoquinones and complexed metal ions. The azino-bis-(3-ethylbenzthiazolin-6-sulfonic acid) cation radical and the ferricenium ion are the best electron acceptors, as judged by the catalytic efficiencies (k(cat)/K(m)). The enzyme may play a role in lignocellulose degradation.     

Expression and functional characterization of P2Y1 and P2Y12 nucleotide receptors in long-term serum-deprived glioma C6 cells

We characterized the expression and functional properties of the ADP-sensitive P2Y(1) and P2Y(12) nucleotide receptors in glioma C6 cells cultured in medium devoid of serum for up to 96 h. During this long-term serum starvation, cell morphology changed from fibroblast-like flat to round, the adhesion pattern changed, cell-cycle arrest was induced, extracellular signal-regulated kinase (ERK1/2) phosphorylation was reduced, Akt phosphorylation was enhanced, and expression of the P2Y(12) receptor relative to P2Y(1) was increased. These processes did not reflect differentiation into astrocytes or oligodendrocytes, as expression of glial fibrillary acidic protein and NG2 proteoglycan (standard markers of glial cell differentiation) was not increased during the serum deprivation. Transfer of the cells into fresh medium containing 10% fetal bovine serum reversed the changes. This demonstrates that serum starvation caused only temporary growth arrest of the glioma C6 cells, which were ready for rapid division as soon as the environment became more favorable. In cells starved for 72 and 96 h, expression of the P2Y(1) receptor was low, and the P2Y(12) receptor was the major player, responsible for ADP-evoked signal transduction. The P2Y(12) receptor activated ERK1/2 kinase phosphorylation (a known cell proliferation regulator) and stimulated Akt activity. These effects were reduced by AR-C69931MX, a specific antagonist of the P2Y(12) receptor. On the other hand, Akt phosphorylation increased in parallel with the low expression of the P2Y(1) receptor, indicating the inhibitory role of P2Y(1) in Akt pathway signaling. The shift in nucleotide receptor expression from P2Y(1) to P2Y(12) would appear to be a new and important self-regulating mechanism that promotes cell growth rather than differentiation and is a defense mechanism against effects of serum deprivation.     

Receiver bias for exaggerated signals in honeybees and its implications for the evolution of floral displays

Mechanistic models of animal signals posit the occurrence of biases on the part of receivers that could be potentially exploited by signallers. Such biases are most obvious when animals are confronted with exaggerated versions of signals they normally encounter. Signalling systems operating in plant-pollinator interactions are among the most highly coevolved, with plants using a variety of floral signals to attract pollinators. A number of observations suggest that pollinators preferentially visit larger floral displays although the benefit of this to either the plant or the pollinator is not always clear. We use a standard dual-choice experimental protocol to show that honeybees display a receiver bias for exaggerated size and colour contrast--two important components of floral signals--even when such signals do not indicate quality. We discuss the implications of this receiver bias for the evolution of floral displays and its possible exploitation by invading alien plants.