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Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

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Molecular design, synthesis and evaluation of chemical biology tools
Hoogenboom, Jorin - \ 2017
Wageningen University. Promotor(en): Han Zuilhof, co-promotor(en): Tom Wennekes. - Wageningen : Wageningen University - ISBN 9789463430388 - 206
chemical biology - biology - tools - synthesis - organic chemistry - molecules - design - chemische biologie - biologie - gereedschappen - synthese - organische scheikunde - moleculen - ontwerp

Chapter 1 provides a perspective of synthetic organic chemistry as a discipline involved in the design, synthesis and evaluation of complex molecules. The reader is introduced with a brief history of synthetic organic chemistry, all the while dealing with different aspects of synthetic organic chemistry. These aspects include design, synthesis and evaluation of complex molecules, which are described with representative examples. For instance, chapter 1 described different strategies to design antibiotics like penicillin. Furthermore, efforts at the interface of chemistry and biology has led to the emerging of a new subdisciplines such as chemical biology. Although this discipline is relatively new, the scientific community has witnessed many breakthrough discoveries and some of these discoveries are highlighted in this chapter. After the general introduction presented in chapter 1, the following chapters of this thesis focus on the design, synthesis and evaluation of small molecular probes towards the study of proteins and glycans in plant and mammalian cells. Most of these probes are complex glycomimetic small molecules that in many instances are prepared with a nitrone-olefin [3+2] cycloaddition as a key step.

In chapter 2, a novel nitrone is presented for the nitrone-olefin [3+2] cycloaddition reaction. This reaction is a powerful tool in synthetic organic chemistry for the synthesis of a wide range of complex molecules. The versatile nature of the reaction is illustrated by the synthesis of several classes of natural product such as vitamins and alkaloids – all complex molecules containing multiple neighboring chiral centers – through the nitrone-olefin [3+2] cycloaddition. However, most nitrones that show good regio- and stereoselectivity are limited in their synthetic versatility, as subsequent synthetic modification of the cycloaddition products are limited. The nitrone described in chapter 2 is a novel masked aldehyde-containing nitrone. This nitrone is prepared by a simple and scalable procedure and can be combined with a diverse set of olefins and other dipolarophiles to afford a broad range of cycloadducts. These cycloadducts can be considered as a masked form of amino-aldehydes, which makes them interesting from a synthetic point of view as illustrated by several postcycloaddition modifications.

The nitrone-olefin [3+2] cycloaddition is also utilized in chapter 3 for the synthesis of glycomimetic building blocks. Glycomimetics such as iminosugars and pipecolic acids are found in nature and possess a variety of biological activities. The potential of glycomimetics have led to the development of drugs for the treatment of diseases such as type 2 diabetes, Gaucher disease and HIV. Chapter 3 describes how glycomimetic building blocks can be obtained through a nitrone-olefin [3+2] cycloaddition, providing different bicyclic isoxazolidines. These cycloadducts are synthetically versatile, as we report a set of reactions that allow selective modification at each functional position. Accordingly, these versatile bicyclic isoxazolidines enable the synthesis of different glycomimetic building blocks. For example, we were able to make a library of pipecolic acid derivatives – a popular drug-motif – via a one-pot Staudinger/aza-Wittig/Ugi three-component reaction.

The bicyclic isoxazolidines, discussed in chapter 3, are also reported in chapter 4. This chapter describes the development of a synthetic route towards an activity-based probe (ABP) to study the enzymatic activity of neuraminidases. Neuraminidases are a class of enzymes found in a range of organisms including mammals. The importance of neuraminidases is illustrated by the existence of a neuraminidase-related genetic disease, sialidosis. With no cure for this fatal disease being very limited, there is keen interest in the discovery of novel mechanisms to restore neuraminidase activity. The poor enzyme-activity of a different enzyme-related disease, Gaucher disease, could be improved through the identification of a small molecule that stabilizes and/or promotes the folding of the active enzyme. The validation of this small molecule was aided greatly by the development of a sensitive ABP targeting this disease specific enzyme. Accordingly, the development of a neuramidase-ABP would provide a diagnostic tool to study the activity of neuramidases and ultimately help identify small molecules that could increase the activity of mutant-neuraminidase molecules by stabilizing and/or promoting the folding of this enzyme. The synthesis of a carbocyclic neuraminidase ABP was the goal of chapter 4 and was approached by starting with the nitrone-olefin [3+2] cycloaddition as the key-step. This reaction provided a bicyclic cycloadduct that was used for the development of a synthetic route, which led to a high yielding and practical synthesis of an advance intermediate possessing the majority of the stereochemistry of the required carbocyclic neuraminidase ABP.

Chapter 5 describes the synthesis and evaluation of chemical tools to study phoshatidyl ethanolamine-binding proteins (PEBPs). This family of proteins is found in a variety of organisms including mammals and plants. This family includes FLOWERING LOCUS T (FT), a signaling protein that acts as a vital flowering hormone in plants. No small molecule inhibitors for FT are known, but an inhibitor called locostatin has been reported to bind in the highly conserved ligand binding site of a structurally related protein. Based on this conservation and overall structurally similarity with FT it was hypothesized that locostatin or derivatives thereof could covalently bind in the ligand binding pocket of FT and hence affect flowering. Chapter 5 reports on the synthesis of novel locostatin-based chemical PEBP probes, followed by evaluation for their ability and selectivity towards FT and a mammalian PEBP.

Chemical tools were also used to study plants in chapter 6, although different aspects of plants were investigated. This chapter describes the direct molecular imaging of carbohydrates (glycans) in Arabidopsis thaliana. Glycans play a crucial but not fully understood role in plant health and development. The formation of glycans is not genetically encoded, which makes it impossible to image them in vivo using genetically encoded fluorescent tags and related molecular biology approaches. A solution to this problem is the use of tailor-made glycans that are metabolically incorporated in plants via the roots, which may then be visualized with copper-catalyzed click labeling. However, this labelling-technique is toxic to plants and future applications would benefit from bio-orthoganol copper-free labeling techniques. Chapter 6 shows, for the first time in metabolic labeling and imaging of plant glycans, the potential of two copper-free click chemistry methods. These methods are bio-orthogonal and lead to more uniform labeling. Furthermore, this chapter also describes the metabolic incorporation of five novel monosaccharide probes in Arabidopsis thaliana roots and their imaging after (copper-free) fluorescent labeling.

Finally, chapter 7 contains a general discussion, critically summarizing the body of this thesis along with additional ideas and recommendations for further research.

Nanoscale force sensors to study supramolecular systems
Cingil, E.H. - \ 2016
Wageningen University. Promotor(en): Martien Cohen Stuart, co-promotor(en): Joris Sprakel. - Wageningen : Wageningen University - ISBN 9789462576971 - 136 p.
sensors - supramolecular chemistry - molecules - biopolymers - polymers - methodology - rheology - supramoleculaire chemie - moleculen - biopolymeren - polymeren - methodologie - reologie

Supramolecular systems are solutions, suspensions or solids, formed by physical and non-covalent interactions. These weak and dynamic bonds drive molecular self-assembly in nature, leading to formation of complex ordered structures in high precision. Understanding self-assembly and co-assembly is crucial to unravel and mimic many processes occurring in nature. However, the challenge cannot be easily addressed especially in biological systems as it involves many dynamic interactions which may cooperatively, noncooperatively or competitively generate a complex manifold of interaction pathways. In this thesis, we employed two techniques to understand these complex interactions in various supramolecular systems at the nanoscale 1) multiple particle tracking microrheology to study thermoreversible assembly of triple helices in a collagen-inspired recombinant polypeptide in the form of a triblock copolymer gel former; and 2) polyfluorene-based conjugated polyelctrolyte mechosensors to monitor electrostatic co-assembly dynamics of (i) a recombinant diblock copolypeptide which encapsulates the conjugated polyelectrolyte like a protein capsid and (ii) various synthetic diblock copolymers which forms complex coacervate micelles; and finally the orthogonal self-assembly dynamics of (iii) a recombinant viral coat protein which mimics natural rod-like viruses. These novel polymeric mechanosensors work as versatile, non-invasive tools to detect even low degrees of analyte binding or complex formation due to the stress applied on their conjugated backbone. This mechanical stress causes the polymeric backbone to stretch which can be detected by a shift in its fluorescence spectra.

Host-interaction effector molecules of Lactobacillus plantarum WCFS1
Lee, I.C. - \ 2016
Wageningen University. Promotor(en): Michiel Kleerebezem, co-promotor(en): P.A. Bron. - Wageningen : Wageningen University - ISBN 9789462576858 - 183 p.
lactobacillus plantarum - molecules - probiotics - immunomodulatory properties - lipoproteins - interactions - molecular interactions - host pathogen interactions - moleculen - probiotica - immunomodulerende eigenschappen - lipoproteïnen - interacties - moleculaire interacties - gastheer-pathogeen interacties

Abstract

Lactobacillus plantarum is found in various environmental habitats, including fermentation products and the mammalian gastrointestinal tract, and specific strains are marketed as probiotics, which are defined as ‘live microorganisms which when administered in adequate amounts confer a health benefit on the host’. Throughout the studies of the mechanisms underlying probiotic activity, it became apparent that the probiotic effects are often species and/or strain specific. This situation has led more researchers to focus on the molecular characteristics of probiotic strains intending to link specific molecular structures to specific probiotic functions, and thereby deduce the mechanisms of molecular communication of probiotics. This thesis focuses on potential cell envelope effector molecules involved in interaction with the mammalian host cells, including lipoteichoic acid (LTA), lipo- and glyco-proteins, and extracellular polysaccharides (EPS), of L. plantarum WCFS1, a model strain for probiotic lactobacilli with a well-annotated genome sequences and sophisticated genetic engineering tools. First, existing research regarding the potential roles in probiotic functionality of Lactobacillus surface molecules in terms of their biosynthesis pathways and structure variations as well as interaction with host Pattern Recognition Receptors (PRRs) and immunomodulatory properties of these molecules are summarized and compared to provide an overview of the state-of-the-art in probiotic effector molecule research. Subsequently, specific molecules that reside in the cell envelope of L. plantarum WCFS1 were study for their role in bacterial physiology, as well as their role as ligands in Toll-like receptor (TLR) 2 signaling and immunomodulatory properties using human-cell co-incubation models. Our results showed that the deficiency of LTA had a drastic impact on cell division, cell morphology and growth in L. plantarum WCFS1, while LTA-deficient cells also elicited more pro-inflammatory responses in PBMCs rather than the expected loss of pro-inflammatory capacity as was observed with similar mutants of Lactobacillus acidophilus NCFM. Further studies on the signaling capacity of the purified LTA from L. plantarum WCFS1 revealed that these molecules are poor TLR2 activators, which is in clear contrast to the highly potent TLR2 stimulatory capacity of LTA obtained from Bacillus subtilis, implying that structural differences of the LTA produced by different bacteria are prominent determinants of their TLR2 signaling capacity and immunomodulatory properties. Lipoproteins of L. plantarum WCFS1 were studied using a derivative strain that is deficient in prolipoprotein diacylglyceryltransferase (Lgt), which transfers acyl chain moieties onto lipoproteins. The lipid moiety was shown to be important for proper anchoring of lipoproteins and TLR1/2 signaling capacity, but did not affect TLR2/6 signaling, suggesting that lipoproteins of L. plantarum WCFS1 are predominantly (if not exclusively) triacylated. The Lgt deficient strain elicited more pro-inflammatory responses in PBMCs as compared to the wild type, indicating that the native lipoproteins could play a role in dampening inflammation upon host-probiotic interaction. In addition, we explored the protein glycosylation machinery in L. plantarum WCFS1, responsible for the glycosylation of the major autolysin (Acm2) of this bacterium, which was previously shown to be O-glycosylated with N-acetylhexosamine conjugates. Using sequence similarity searches in combination with a lectin-based glycan detection and mass spectrometry analysis, two glycosyl-transferases, GtfA and GtfB (formerly annotated as TagE5 and TagE6, respectively), were shown to be required for the glycosylation of Acm2 and other unidentified L. plantarum WCFS1 glycosylated proteins. These results provide the first example of a general protein-glycosylation machinery in a Lactobacillus species. Finally, extracellular polysaccharides (EPS) in L. plantarum were studied in two strains that produce large amounts of EPS: L. plantarum SF2A35B and Lp90, in comparison to the lowly producing model strain WCFS1. Based on genome sequence comparison, both of the high producer strains were found to possess strain-specific and unique polysaccharide gene clusters. These gene clusters were deleted and the mutants were shown to have lost the capacity to produce large amounts of EPS, and were studied in relation to their properties in host-bacteria interaction. The results illustrate strain-specific and variable impacts of the removal of the EPS in the background of individual L. plantarum strains, supporting the importance of EPS in L. plantarum strains as a strain-specific determinant in host interaction. Overall, this thesis showed that surface molecules not only play important roles in bacterial physiology, but also in the interaction with the host mucosa through pattern recognition receptors expressed by the host cells. With the growing amount of evidence of structural variations in surface molecules, which are influenced by genetic background, physiological status, environmental factors, and other biological processes, these molecules form a unique signature associated with each strain that as a consequence elicits a strain-specific response when interacting with host cells.

Monitoring protein capsid assembly with a conjugated polymer strain sensor
Cingil, E.H. ; Storm, I.M. ; Yorulmaz, Y. ; Brake, D.W. te; Vries, R.J. de; Cohen Stuart, M.A. ; Sprakel, J.H.B. - \ 2015
Journal of the American Chemical Society 137 (2015)31. - ISSN 0002-7863 - p. 9800 - 9803.
beta-phase formation - polyfluorene - poly(9,9-dioctylfluorene) - morphology - photophysics - copolymers - molecules - dynamics - length
Semiconducting polymers owe their optoelectronic properties to the delocalized electronic structure along their conjugated backbone. Their spectral features are therefore uniquely sensitive to the conformation of the polymer, where mechanical stretching of the chain leads to distinct vibronic shifts. Here we demonstrate how the optomechanical response of conjugated polyelectrolytes can be used to detect their encapsulation in a protein capsid. Coating of the sensor polymers by recombinant coat proteins induces their stretching due to steric hindrance between the proteins. The resulting mechanical planarizations lead to pronounced shifts in the vibronic spectra, from which the process of capsid formation can be directly quantified. These results show how the coupling between vibronic states and mechanical stresses inherent to conjugated polymers can be used to noninvasively measure strains at the nanoscale.
De bouwstenen van het leven : een introductie tot de moleculaire celbiologie
Prinsen, J.A.M.M. ; Leij, F.R. van der - \ 2014
Wageningen : Wageningen Academic Publishers - ISBN 9789086862412 - 470
cellen - moleculaire biologie - celbiologie - atomen - moleculen - eiwitten - dna - rna - metabolisme - celdeling - studieboeken - cells - molecular biology - cellular biology - atoms - molecules - proteins - metabolism - cell division - textbooks
In De bouwstenen van het leven wordt - ook voor leken - op een toegankelijke wijze een overzicht gegeven van de basale kennis van de biochemie en de moleculaire celbiologie (ook wel Life Sciences genoemd). De wereld van DNA, RNA en eiwitten, van energiemetabolisme tot en met genetica, is door Jan Prinsen en Feike van der Leij op een overzichtelijke manier begrijpelijk gemaakt. Niet alleen voor Bachelor-studenten een goed alternatief naast Engelstalige standaardwerken, maar voor iedereen die wil weten hoe de processen in ons lichaam en in de natuur om ons heen op moleculair niveau verklaard kunnen worden. De auteurs hebben daarbij een klassieke kijk gecombineerd met de laatste inzichten en maken op een heldere manier gebruik van de soms onvermijdelijke vaktaal. Het is daarmee oprecht een introductie tot de moleculaire celbiologie te noemen. De informatie wordt in lagen aangereikt: de lezer kan zelf de mate van diepgang en detail bepalen.
A detailed comparative study between chemical and bioactive properties of Ganoderma lucidum from different origins
Stojkovic, D.S. ; Barros, L. ; Calhelha, R.C. ; Glamoclija, J. ; Ciric, A. ; Griensven, L.J.L.D. van; Sokovic, M. ; Ferreira, I.C.F.R. - \ 2014
International Journal of Food Sciences and Nutrition 65 (2014)1. - ISSN 0963-7486 - p. 42 - 47.
medicinal mushrooms - antioxidant properties - wild mushrooms - liquid-chromatography - fruiting body - fr. karst - portugal - polysaccharides - molecules - nutrients
A detailed comparative study on chemical and bioactive properties of wild and cultivated Ganoderma lucidum from Serbia (GS) and China (GCN) was performed. This species was chosen because of its worldwide use as medicinal mushroom. Higher amounts of sugars were found in GS, while higher amounts of organic acids were recorded in GCN. Unsaturated fatty acids predominated over saturated fatty acids. GCN revealed higher antioxidant activity, while GS exhibited inhibitory potential against human breast and cervical carcinoma cell lines. No cytotoxicity in non-tumour liver primary cell culture was observed for the different samples. Both samples possessed antibacterial and antifungal activities, in some cases even better than the standard antimicrobial drugs. This is the first study reporting a comparison of chemical compounds and bioactivity of G. lucidum samples from different origins.
Constructing a mass measurement error surface to improve automatic annotations in liquid chromatography/mass spectrometry based metabolomics
Shahaf, N. ; Franceschi, P. ; Arapitsas, P. ; Rogachev, I. ; Vrhovsek, U. ; Wehrens, H.R.M.J. - \ 2013
Rapid Communications in Mass Spectrometry 27 (2013)21. - ISSN 0951-4198 - p. 2425 - 2431.
accuracy - database - recalibration - precision - molecules - sample
RATIONALE Estimation of mass measurement accuracy is an elementary step in the application of mass spectroscopy (MS) data towards metabolite annotations and has been addressed several times in the past. However, the reproducibility of mass measurements over a diverse set of analytes and in variable operating conditions, which are common in high-throughput metabolomics studies, has, to the best of our knowledge, not been addressed so far. METHODS A method to automatically extract mass measurement errors from a large data set of measurements made on a quadrupole time-of-flight (QTOF) MS instrument has been developed. The size of the data processed in this study has enabled us to use a statistical data driven approach to build a model which reliably predicts the confidence interval of the absolute mass measurement error based on individual ion peak conditions in a fast, high-throughput manner. RESULTS We show that our model predictions are reproducible in external datasets generated in similar, but not identical conditions, and have demonstrated the advantage of our approach over the common practice of fixed mass measurement error limits. CONCLUSIONS Outlined is an approach which can promote a more rational use of MS technology by automatically evaluating the absolute mass measurement error based on the individual peak conditions. The immediate application of our method is integration in high-throughput peak annotation pipelines for database searches.
Accurate pKa Calculation of the Conjugate Acids of Alkanolamines, Alkaloids and Nucleotide Bases by Quantum Chemical Methods
Gangarapu, S. ; Marcelis, A.T.M. ; Zuilhof, H. - \ 2013
ChemPhysChem 14 (2013)5. - ISSN 1439-4235 - p. 990 - 995.
density-functional theory - co2 capture technology - gas-phase basicities - pk(a) values - continuum model - free-energy - amines - molecules - prediction - proton
The pKa of the conjugate acids of alkanolamines, neurotransmitters, alkaloid drugs and nucleotide bases are calculated with density functional methods (B3LYP, M08-HX and M11-L) and ab initio methods (SCS-MP2, G3). Implicit solvent effects are included with a conductor-like polarizable continuum model (CPCM) and universal solvation models (SMD, SM8). G3, SCS-MP2 and M11-L methods coupled with SMD and SM8 solvation models perform well for alkanolamines with mean unsigned errors below 0.20 pKa units, in all cases. Extending this method to the pKa calculation of 35 nitrogen-containing compounds spanning 12 pKa units showed an excellent correlation between experimental and computational pKa values of these 35 amines with the computationally low-cost SM8/M11-L density functional approach.
Solvated protein-protein docking using Kyte-Doolittle-based water preferences
Kastritis, P.L. ; Visscher, K.M. ; Dijk, A.D.J. van; Bonvin, A.M.J.J. - \ 2013
Proteins : Structure, Function, and Bioinformatics 81 (2013)3. - ISSN 0887-3585 - p. 510 - 518.
ligand docking - biomolecular complexes - globular-proteins - drug design - molecules - recognition - interfaces - solvent - haddock - challenges
HADDOCK is one of the few docking programs that can explicitly account for water molecules in the docking process. Its solvated docking protocol starts from hydrated molecules and a fraction of the resulting interfacial waters is subsequently removed in a biased Monte Carlo procedure based on water-mediated contact probabilities. The latter were derived from an analysis of water contact frequencies from high-resolution crystal structures. Here, we introduce a simple water mediated amino acid - amino acid contact probability scale derived from the Kyte-Doolittle hydrophobicity scale and assess its performance on the largest high-resolution dataset developed to date for solvated docking. Both scales yield high-quality docking results. The novel and simple hydrophobicity scale, which should reflect better the physico-chemical principles underlying contact propensities, leads to a performance improvement of around 10% in ranking, cluster quality and water recovery at the interface compared to the statistics-based original solvated docking protocol.
Improving the Capture of Co2 by Substituted Monoethanolamines: Electronic Effects of Fluorine and Methyl Substituents
Gangarapu, S. ; Marcelis, A.T.M. ; Zuilhof, H. - \ 2012
ChemPhysChem 13 (2012)17. - ISSN 1439-4235 - p. 3973 - 3980.
main-group thermochemistry - free-energy perturbations - gas-phase basicities - ab-initio - noncovalent interactions - carbon-dioxide - amines - molecules - absorption - accuracy
The influence of electronic and steric effects on the reaction between CO(2) and monoethanolamine (MEA) absorbents is investigated using computational methods. The pK(a) of the alkanolamine, the reaction enthalpy for carbamate formation, and the hydrolytic carbamate stability are important factors for the efficiency of CO(2) capture. The steric and electronic effects of CH(3), CH(2)F, CHF(2), CF(3), F, dimethyl, difluoro, and bis(2-trifluoromethyl) substituents at the a carbon of MEA on this reaction are investigated. Density functional theory (DFT) (B3LYP, M06-2X, M08-HX and M11-L) and ab initio methods [spin component-scaled second-order Møller-Plesset theory (SCS-MP2), G3], each coupled with solvent models [conductor-like polarizable continuum model (CPCM) and universal solvation models (SM8 and SMD)], are shown to yield accurately calculated pK(a) values of the substituted MEAs. Specifically, G3, SCS-MP2, and M11-L methods coupled with the SMD and SM8 solvation models perform well with a mean unsigned error (MUE) of only 0.15, 0.24 and 0.25 pK(a) units, respectively. SCS-MP2 is used to calculate the reaction enthalpy for carbamate formation and the carbamate stability towards hydrolysis. With the introduction of ß-fluoro substituents (especially the CH(2) F moiety) the reaction enthalpy for the formation of carbamates can be fine-tuned to be less exothermic than that using the unsubstituted MEA. This implies a reduced energy requirement for the solvent-regeneration step in the post-combustion carbon-capture method, which is currently the energy-limiting step in efficient CO(2) capture. ß-Fluoro-substituted MEAs are also shown to form less stable carbamates than MEA. Thus, ß-fluoro-substituted MEAs display a great potential for the use in the post-combustion carbon-capture process. Finally, a clear correlation is observed between the gas-phase basicity and the tendency to form carbamates. This allows for the rapid prediction of which species will be formed experimentally, and thus the CO(2)-absorbing capacities of alkanolamines can be estimated
Kleine deeltjes, grote kansen: nanotechnologie neemt een grote vlucht (Interview met Maarten Jongsma)
Smit, A. ; Jongsma, M.A. - \ 2012
WageningenWorld 4 (2012). - ISSN 2210-7908 - p. 34 - 39.
nanotechnologie - bionanotechnologie - smaak - moleculen - organische scheikunde - toegepast onderzoek - nanotechnology - bionanotechnology - taste - molecules - organic chemistry - applied research
Een elektronische tong, zeefjes die binnen een uur ziekmakende bacteriën detecteren, of moleculen die helpen bij het vinden en doden van tumoren. Wageningen UR timmert hard aan de weg met nanotechnologie – en onderzoekt meteen de mogelijke risico’s voor mens en natuur.
The major secreted protein Msp1/p75 is O-glycosylated in Lactobacillus rhamnosus GG
Lebeer, S. ; Claes, I.J. ; Balog, C.I. ; Schoofs, G. ; Verhoeven, T.L.A. ; Nys, K. ; Ossowski, I. von; Vos, W.M. de - \ 2012
Microbial Cell Factories 11 (2012)15. - ISSN 1475-2859
functional-analysis - glycoproteins - biosynthesis - molecules - bacteria - host - pathogens - cell
BACKGROUND: Although the occurrence, biosynthesis and possible functions of glycoproteins are increasingly documented for pathogens, glycoproteins are not yet widely described in probiotic bacteria. Nevertheless, knowledge of protein glycosylation holds important potential for better understanding specific glycan-mediated interactions of probiotics and for glycoengineering in food-grade microbes. RESULTS: Here, we provide evidence that the major secreted protein Msp1/p75 of the probiotic Lactobacillus rhamnosus GG is glycosylated. Msp1 was shown to stain positive with periodic-acid Schiff staining, to be susceptible to chemical deglycosylation, and to bind with the mannose-specific Concanavalin A (ConA) lectin. Recombinant expression in Escherichia coli resulted in a significant reduction in molecular mass, loss of ConA reactivity and increased sensitivity towards pronase E and proteinase K. Mass spectrometry showed that Msp1 is O-glycosylated and identified a glycopeptide TVETPSSA (amino acids 101-108) bearing hexoses presumably linked to the serine residues. Interestingly, these serine residues are not present in the homologous protein of several Lactobacillus casei strains tested, which also did not bind to ConA. The role of the glycan substitutions in known functions of Msp1 was also investigated. Glycosylation did not seem to impact significantly on the peptidoglycan hydrolase activity of Msp1. In addition, the glycan chain appeared not to be required for the activation of Akt signaling in intestinal epithelial cells by Msp1. On the other hand, examination of different cell extracts showed that Msp1 is a glycosylated protein in the supernatant, but not in the cell wall and cytosol fraction, suggesting a link between glycosylation and secretion of this protein. CONCLUSIONS: In this study we have provided the first evidence of protein O-glycosylation in the probiotic L rhamnosus GG. The major secreted protein Msp1 is glycosylated with ConA reactive sugars at the serine residues at 106 and 107. Glycosylation is not required for the peptidoglycan hydrolase activity of Msp1 nor for Akt activation capacity in epithelial cells, but appears to be important for its stability and protection against proteases
Fluorescence of Alexa Fluor dye tracks protein folding
Lindhoud, S. ; Westphal, A.H. ; Borst, J.W. ; Visser, A.J.W.G. ; Mierlo, C.P.M. van - \ 2012
PLoS ONE 7 (2012)10. - ISSN 1932-6203 - 8 p.
azotobacter-vinelandii apoflavodoxin - resonance energy-transfer - beta parallel protein - molten-globule state - flavodoxin-ii - molecules - pathway - chains - intermediate - spectroscopy
Fluorescence spectroscopy is an important tool for the characterization of protein folding. Often, a protein is labeled with appropriate fluorescent donor and acceptor probes and folding-induced changes in Förster Resonance Energy Transfer (FRET) are monitored. However, conformational changes of the protein potentially affect fluorescence properties of both probes, thereby profoundly complicating interpretation of FRET data. In this study, we assess the effects protein folding has on fluorescence properties of Alexa Fluor 488 (A488), which is commonly used as FRET donor. Here, A488 is covalently attached to Cys69 of apoflavodoxin from Azotobacter vinelandii. Although coupling of A488 slightly destabilizes apoflavodoxin, the three-state folding of this protein, which involves a molten globule intermediate, is unaffected. Upon folding of apoflavodoxin, fluorescence emission intensity of A488 changes significantly. To illuminate the molecular sources of this alteration, we applied steady state and time-resolved fluorescence techniques. The results obtained show that tryptophans cause folding-induced changes in quenching of Alexa dye. Compared to unfolded protein, static quenching of A488 is increased in the molten globule. Upon populating the native state both static and dynamic quenching of A488 decrease considerably. We show that fluorescence quenching of Alexa Fluor dyes is a sensitive reporter of conformational changes during protein folding.
Effect of natural and chimeric haemagglutinin genes on influenza A virus replication in baby hamster kidney cells.
Wielink, R. van; Harmsen, M.M. ; Martens, D.E. ; Leeuw, O.S. de; Peeters, B.P.H. ; Wijffels, R.H. - \ 2012
Journal of Biotechnology 162 (2012)2-3. - ISSN 0168-1656 - p. 197 - 201.
candidate vaccine viruses - system - generation - molecules - growth - lines
Baby hamster kidney (BHK21) cells are used to produce vaccines against various viral veterinary diseases, including rabies and foot-and-mouth-disease. Although particular influenza virus strains replicate efficiently in BHK21 cells the general use of these cells for influenza vaccine production is prohibited by the poor replication of most strains, including model strain A/PR/8/34 [H1N1] (PR8). We now show that in contrast to PR8, the related strain A/WSN/33 [H1N1] (WSN) replicates efficiently in BHK21 cells. This difference is determined by the haemagglutinin (HA) protein since reciprocal reassortant viruses with swapped HAs behave similarly with respect to growth on BHK21 cells as the parental virus from which their HA gene is derived. The ability or inability of six other influenza virus strains to grow on BHK21 cells appears to be similarly dependent on the nature of the HA gene since reassortant PR8 viruses containing the HA of these strains grow to similar titres as the parental virus from which the HA gene was derived. However, the growth to low titres of a seventh influenza strain was not due to the nature of the HA gene since a reassortant PR8 virus containing this HA grew efficiently on BHK21 cells. Taken together, these results suggest that the HA gene often primarily determines influenza replication efficiency on BHK21 cells but that in some strains other genes are also involved. High virus titres could be obtained with reassortant PR8 strains that contained a chimeric HA consisting of the HA1 domain of PR8 and the HA2 domain of WSN. HA1 contains most antigenic sites and is therefore important for vaccine efficacy. This method of producing the HA1 domain as fusion to a heterologous HA2 domain could possibly also be used for the production of HA1 domains of other viruses to enable the use of BHK21 cells as a generic platform for veterinary influenza vaccine production.
Functional Analysis of Lactobacillus rhamnosus GG Pili in Relation to Adhesion and Immunomodulatory Interactions with Intestinal Epithelial Cells
Lebeer, S. ; Claes, I.J. ; Tytgat, H.L.P. ; Verhoeven, T.L.A. ; Marien, E. ; Ossowski, I. von; Reunanen, J. ; Palva, A. ; Vos, W.M. de; Keersmaecker, S.C. de; Vanderleyden, J. - \ 2012
Applied and Environmental Microbiology 78 (2012)1. - ISSN 0099-2240 - p. 185 - 193.
gram-positive bacteria - gene-expression - salmonella-typhimurium - lipoteichoic acid - biofilm formation - reveals - protein - inflammation - molecules - pathogens
Lactobacillus rhamnosus GG, a probiotic with good survival capacity in the human gut, has well-documented adhesion properties and health effects. Recently, spaCBA-encoded pili that bind to human intestinal mucus were identified on its cell surface. Here, we report on the phenotypic analysis of a spaCBA pilus knockout mutant in comparison with the wild type and other adhesin mutants. The SpaCBA pilus of L. rhamnosus GG showed to be key for efficient adherence to the Caco-2 intestinal epithelial cell (IEC) line and biofilm formation. Moreover, the spaCBA mutant induces an elevated level of interleukin-8 (IL-8) mRNA in Caco-2 cells compared to the wild type, possibly involving an interaction of lipoteichoic acid with Toll-like receptor 2. In contrast, an L. rhamnosus GG mutant without exopolysaccharides but with an increased exposure of pili leads to the reduced expression of IL-8. Using Transwells to partition bacteria from Caco-2 cells, IL-8 induction is blocked completely regardless of whether wild-type or mutant L. rhamnosus GG cells are used. Taken together, our data suggest that L. rhamnosus GG SpaCBA pili, while promoting strong adhesive interactions with IECs, have a functional role in balancing IL-8 mRNA expression induced by surface molecules such as lipoteichoic acid
Kinetic and Stoichiometric Characterisation of Streptavidin-Binding Aptamers
Ruigrok, V.J.B. ; Duijn, E. van; Barendregt, A. ; Dyer, K. ; Tainer, J.A. ; Stoltenburg, R. ; Strehlitz, B. ; Levisson, M. ; Smidt, H. ; Oost, J. van der - \ 2012
ChemBioChem 13 (2012)6. - ISSN 1439-4227 - p. 829 - 836.
scattering data-analysis - in-vitro selection - dna aptamers - web server - ligands - molecules - evolution - saxs
Aptamers are oligonucleotide ligands that are selected for high-affinity binding to molecular targets. Only limited knowledge relating to relations between structural and kinetic properties that define aptamer-target interactions is available. To this end, streptavidin-binding aptamers were isolated and characterised by distinct analytical techniques. Binding kinetics of five broadly similar aptamers were determined by surface plasmon resonance (SPR); affinities ranged from 35-375 nM with large differences in association and dissociation rates. Native mass spectrometry showed that streptavidin can accommodate up to two aptamer units. In a 3D model of one aptamer, conserved regions are exposed, strongly suggesting that they directly interact with the biotin-binding pockets of streptavidin. Mutational studies confirmed both conserved regions to be crucial for binding. An important result is the observation that the most abundant aptamer in our selections is not the tightest binder, emphasising the importance of having insight into the kinetics of complex formation. To find the tightest binder it might be better to perform fewer selection rounds and to focus on post-selection characterisation, through the use of complementary approaches as described in this study
Influence of PEGylation with linear and branched PEG chains on the adsorption of glucagon to hydrophobic surfaces
Pinholt, C. ; Bukrinsky, J.T. ; Hostrup, S. ; Frokjaer, S. ; Norde, W. ; Jorgensen, L. - \ 2011
European Journal of Pharmaceutics and Biopharmaceutics 77 (2011)1. - ISSN 0939-6411 - p. 139 - 147.
poly(ethylene glycol)-modified lysozyme - protein adsorption - polyethylene-glycols - silica - conformation - fluorescence - association - molecules - fragments - peptide
PEGylation has proven useful for prolonging the plasma half lives of proteins, and since approval of the first PEGylated protein drug product by the FDA in 1990, several PEGylated protein drug products have been marketed. However, the influence of PEGylation on the behavior of proteins at interfaces is only poorly understood. The aim of this work was to study the effect of PEGylation on the adsorption of glucagon from aqueous solution to a hydrophobic surface and to compare the effects of PEGylation with a linear and a branched PEG chain, respectively. The 3483 Da peptide glucagon was PEGylated with a 2.2 kDa linear and a branched PEG chain, respectively, and the adsorption behaviors of the three proteins were compared using isothermal titration calorimetry, fixed-angle optical reflectometry and total internal reflection fluorescence. PEGylation decreased the number of glucagon molecules adsorbing per unit surface area and increased the initial adsorption rate of glucagon. Furthermore, the results indicated that the orientation and/or structural changes of glucagon upon adsorption were affected by the PEGylation. Finally, from the isothermal titration calorimetry and the reflectometry data, it was observed that the architecture of the PEG chains had an influence on the observed heat flow upon adsorption as well as on the initial rate of adsorption, respectively.
A novel soluble immune-type receptor (SITR) in teleost fish: carp SITR is involved in the nitric oxide-mediated response to a protozoan parasite
Ribeiro, C.M.S. ; Raes, G. ; Ghassabeh, G.H. ; Schijns, V.E.J.C. ; Pontes, M.J.S.L. ; Savelkoul, H.F.J. ; Wiegertjes, G.F. - \ 2011
PLoS ONE 6 (2011)1. - ISSN 1932-6203 - 20 p.
immunoglobulin-like transcripts - ig-like domains - cyprinus-carpio - trypanoplasma-borreli - gene-cluster - monoclonal-antibodies - natural cytotoxicity - evolution - molecules - recognition
Background- The innate immune system relies upon a wide range of germ-line encoded receptors including a large number of immunoglobulin superfamily (IgSF) receptors. Different Ig-like immune receptor families have been reported in mammals, birds, amphibians and fish. Most innate immune receptors of the IgSF are type I transmembrane proteins containing one or more extracellular Ig-like domains and their regulation of effector functions is mediated intracellularly by distinct stimulatory or inhibitory pathways. Methodology/Principal Findings - Carp SITR was found in a substracted cDNA repertoire from carp macrophages, enriched for genes up-regulated in response to the protozoan parasite Trypanoplasma borreli. Carp SITR is a type I protein with two extracellular Ig domains in a unique organisation of a N-proximal V/C2 (or I-) type and a C-proximal V-type Ig domain, devoid of a transmembrane domain or any intracytoplasmic signalling motif. The carp SITR C-proximal V-type Ig domain, in particular, has a close sequence similarity and conserved structural characteristics to the mammalian CD300 molecules. By generating an anti-SITR antibody we could show that SITR protein expression was restricted to cells of the myeloid lineage. Carp SITR is abundantly expressed in macrophages and is secreted upon in vitro stimulation with the protozoan parasite T. borreli. Secretion of SITR protein during in vivo T. borreli infection suggests a role for this IgSF receptor in the host response to this protozoan parasite. Overexpression of carp SITR in mouse macrophages and knock-down of SITR protein expression in carp macrophages, using morpholino antisense technology, provided evidence for the involvement of carp SITR in the parasite-induced NO production. Conclusion/Significance - We report the structural and functional characterization of a novel soluble immune-type receptor (SITR) in a teleost fish and propose a role for carp SITR in the NO-mediated response to a protozoan parasite.
Comparison of various models to describe the charge-pH dependence of poly(acrylic acid)
Lützenkirchen, J. ; Male, J. van; Leermakers, F.A.M. ; Sjöberg, S. - \ 2011
Journal of Chemical and Engineering Data 56 (2011)4. - ISSN 0021-9568 - p. 1602 - 1612.
ion-binding - electrolyte-solutions - monte-carlo - polyelectrolytes - adsorption - molecules - constants - titration - sodium - simulations
The charge of poly(acrylic acid) (PAA) in dilute aqueous solutions depends on pH and ionic strength. We report new experimental data and test various models to describe the deprotonation of PAA in three different NaCl concentrations. A simple surface complexation approach is found to be very successful: the constant capacitance model requires one pKa value and one capacitance for excellent fits to the data, with both parameters depending on ionic strength. The use of a self-consistent set of diffuse double layer parameters with one pKa for flat, spherical, and cylindrical geometry does not result in a satisfactory description of the data, and a number of adjustments to that model were tested to improve the fit. The basic Stern model (BSM) was tested with both plate and cylinder geometry. The cylinder geometry along with strong electrolyte binding was found to be superior to a similar approach involving weak electrolyte binding both in terms of goodness of fit and self-consistency of the parameters. The third approach, the non-ideal competitive consistent adsorption-Donnan (NICCA-Donnan) model, involving one functional group, allows an excellent description of the experimental data. Finally, the polyacid chain was modeled using a mechanistically more realistic self-consistent field (SCF) approach, which allows for radially inhomogeneous distributions of the charges and radial variations in the polymer density and electrostatic potential, while the functional groups can be in protonated, deprotonated, or complexed states. One functional group was insufficient for a satisfactory description of the data. With two segments (one monoprotic, the other diprotic) a reasonable description of the data, including the ionic strength dependence, is achieved, and the tendency of the size of the macro-ion with pH and ionic strength is as expected. This model has the fewest adjustable parameters and is considered the most realistic and comprehensive among the models tested
Gene expression patterns in the ventral tegmental area relate to oestrus behaviour in high-producing dairy cows
Wyszynska-Koko, J. ; Wit, A.A.C. de; Beerda, B. ; Veerkamp, R.F. ; Pas, M.F.W. te - \ 2011
Journal of Animal Breeding and Genetics 128 (2011)3. - ISSN 0931-2668 - p. 183 - 191.
cerebral-cortex - immunoglobulin - fertility - normalization - microarray - molecules - profiles - adhesion - neurons - stress
Reduced oestrus behaviour expression or its absence (silent oestrus) results in subfertility in high-producing dairy cows. Insight into the genomic regulation of oestrus behaviour is likely to help alleviate reproduction problems. Here, gene expression was recorded in the ventral tegmental area (VTA) of high milk production dairy cows differing in the degree of showing oestrus behaviour (H – highly expressing versus L – lowly expressing), which was then analysed. Genes regulating cell morphology and adhesion or coding for immunoglobulin G (IgG) chains were differentially expressed in VTA between cows around day 0 and 12 of the oestrus cycle, but only in cows that earlier in life tended to show high levels of oestrus behaviour (H0 versus H12). The comparisons between H and L groups of cows also revealed differential expression of several genes (e.g. those of the IgG family or encoding for pro-melanin-concentrating hormone). However, any significant changes in VTA genes expression were detected in the comparison of L0 versus L12 cows. Altogether, the genes expression profile in VTA of cows highly expressing oestrus behaviour changes together with phases of the oestrus cycle, while in case of cows expressing oestrus behaviour lowly it remains stable. This supports the existence of genomic regulation by centrally expressed genes on the expression of oestrus behaviour in dairy cows.
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