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Enrichment proteomics challenges and perspectives : analysis of the N-glycoproteome and plasma membrane proteome in glycosylation mutants and plant-pathogen interactions
Song, Wei - \ 2016
Wageningen University. Promotor(en): Harro Bouwmeester, co-promotor(en): Sander van der Krol; Twan America. - Wageningen : Wageningen University - ISBN 9789462578722 - 172
proteomics - glycoproteins - arabidopsis - plant-animal interactions - plant pathogens - plasma membranes - eiwitexpressieanalyse - glycoproteïnen - plant-dier interacties - plantenziekteverwekkers - plasmamembranen
This thesis is based on two technology projects from the Centre for BioSystems Genomics (CBSG), entitled ‘Comparative proteomics on Plant Pathogen interactions through enrichment of the N-glycoproteome and tagged-glycoproteome’ (TD7) and ‘Plasma Membrane proteomics for Plant Pathogen interactions’ (TD5). In the former project we developed the protocol for isolation, identification and quantification of N-linked glycoproteins from plants and used it to obtain a comprehensive inventory of glycan-occupancy of Arabidopsis glycoproteins. In the second project, a protocol for the enrichment of plasma membrane (PM) fraction from plant material was developed and applied to study the role of the PM proteome in the interaction of plants with the plant pathogen Phytophthora infestans. Combined these activities have resulted in a thesis devoted to technical developments in label-free comparative enrichment proteomics, with validation in a number of different biological systems.
Plant Biotechnology meets Immunology : plant-based expression of immunologically relevant proteins
Wilbers, R.H.P. - \ 2015
Wageningen University. Promotor(en): Jaap Bakker, co-promotor(en): Arjen Schots; Geert Smant. - Wageningen : Wageningen University - ISBN 9789462574335 - 229
plantenbiotechnologie - immunologie - planten - eiwitten - farmaceutische eiwitten - interleukine 10 - ontstekingsremmers - biologische activiteit - cytokinen - genexpressie - transforming growth factor - wormen - recombinant eiwitten - glycoproteïnen - plant biotechnology - immunology - plants - proteins - pharmaceutical proteins - interleukin 10 - antiinflammatory agents - biological activity - cytokines - gene expression - helminths - recombinant proteins - glycoproteins
The incidence of inflammatory disorders in industrialized countries has dramatically increased over the last decennia, which is believed to result from a change in life-style. Treatment of these inflammatory disorders relies on the intervention in immune responses thereby restoring homeostasis. For now, many inflammatory disorders are treated with broad-acting immunosuppressive drugs or monoclonal antibodies that specifically target pro-inflammatory molecules of the immune system. An alternative therapeutic approach would be to use immunomodulatory proteins that are naturally involved in re-establishing immune homeostasis. This thesis describes the plant-based expression of a variety of immunomodulatory cytokines that may be used as biopharmaceutical proteins in the future. Furthermore, this thesis contains a pioneering chapter on the plant-based expression of immunomodulatory helminth-secreted glycoproteins.
In Chapter 2 we describe the plant-based expression of the immune-regulatory cytokine human transforming growth factor β1 (TGF-β1). By co-expressing human furin with latent TGF-β1 we were able to engineer the post-translational proteolytic processing of TGF-β1, which enabled the production of biologically active TGF-β1. In Chapter 3 we reveal that aggregation is a major production bottleneck for the anti-inflammatory cytokine interleukin-10 (IL-10). By protein engineering we were able to prevent aggregation and created a biologically active fusion protein of IL-10. In Chapter 4 we express biologically active IL-22 in plants. We reveal that, in contrast to current literature, its activity is independent of the presence of N-glycans or their composition. This chapter further reveals that plants offer a powerful tool to allow investigation into the role of N-glycans in protein folding and biological activity of glycoproteins. In Chapter 5 we further explore the potential of glyco-engineering in plants by engineering helminth-like N-glycans. We produce large quantities of two major egg antigens from Schistosoma mansoni and successfully engineer Lewis X, LDN and LDNF N-glycan structures. These plant biotechnological research lines are a showcase for the potential of engineering proteins as well as post-translational modifications in plants with special emphasis on N-glycan engineering. Altogether, the results presented in the first four chapters reveal the remarkable flexibility of plants as a production platform for recombinant proteins. It showcases the potential of engineering proteins as well as post-translational modifications in plants, but it especially highlights the engineering of tailor made N-glycans in plants. This, combined with the speed of transient expression by means of agroinfiltration, makes transient expression in Nicotiana benthamiana a powerful tool to study the role of N-glycans on glycoprotein function.
In parallel to these plant biotechnological research lines, we also developed an in vitro model system based on mouse bone marrow-derived cells to study immunological responses. We used this model to obtain clues on why IL-10 therapy has not been as successful as previously anticipated. In Chapter 6 we have set-up biological activity assays based on bone marrow-derived cells and reveal that IL-10 activity is dependent on both IL-10R1 and IL-10R2, but not IL-10R2-associated signalling via Tyk2. We also show that interactions between IL-10R1 and IL-10R2 (both intracellular and extracellular) reduce cellular binding of IL-10, but are crucial to initiate IL-10 mediated signalling. Furthermore, we observed that macrophages and dendritic cells respond differently to IL-10. This was further investigated in Chapter 7 where we reveal that GM-CSF (the cytokine used to differentiate dendritic cells) is responsible for negatively regulating early IL-10-mediated responses. Strikingly, GM-CSF does not strongly affect the IL-10-induced activation of the transcription factor STAT3. Instead, GM-CSF induces strong constitutive phosphorylation of GSK-3β, a signalling component downstream of the PI3K/Akt pathway. These immunological chapters give novel insights on the mechanism of initiating IL-10-induced signalling and on the possible integration of signal transduction pathways elicited by different cytokines. Ultimately this knowledge could provide us with new therapeutic strategies to treat inflammatory disorders.
Label-Free Glycoprofiling with Multiplex Surface Plasmon Resonance: A Tool To Quantify Sialylation of Erythropoietin
Geuijen, K.P.M. ; Halims, L.A. ; Schellekens, H. ; Schasfoort, R.B. ; Wijffels, R.H. ; Eppink, M.H.M. - \ 2015
Analytical Chemistry 87 (2015)16. - ISSN 0003-2700 - p. 8115 - 8122.
liquid chromatography/mass spectrometry - recombinant-human-erythropoietin - lectin microarray - biological function - real-time - cho-cells - glycosylation - carbohydrate - glycoproteins - hemoglobin
Protein glycosylation is among the most common and well-defined post-translational modifications due to its vital role in protein function. Monitoring variation in glycosylation is necessary for producing more effective therapeutic proteins. Glycans attached to glycoproteins interact highly specific with lectins, natural carbohydrate-binding proteins, which property is used in the current label-free methodology. We have established a lectin microarray for label-free detection of lectin-carbohydrate interactions allowing us to study protein glycosylation directly on unmodified glycoproteins. The method enables simultaneous measurement of up to 96 lectin-carbohydrate interactions on a multiplex surface plasmon resonance imaging platform within 20 min. Specificity determination of lectins succeeded by analysis of neoglycoproteins and enzymatically remodeled glycoproteins to verify carbohydrate binding. We demonstrated the possibilities for glycosylation fingerprinting by comparing different Erythropoietin sources without the need for any sample pretreatment and we were able to accurately quantify relative sialylation levels of Erythropoietin.
Expression of natural human b1,4-GalT1 variants and of non-mammalian homologues in plants leads to differences in galactosylation of N-glycans
Hesselink, T. ; Rouwendal, G.J.A. ; Henquet, M.G.L. ; Florack, D.E.A. ; Helsper, J.P.F.G. ; Bosch, H.J. - \ 2014
Transgenic Research 23 (2014)5. - ISSN 0962-8819 - p. 717 - 728.
golgi-apparatus - murine beta-1,4-galactosyltransferase - beta 1,4-galactosyltransferase - transgenic plants - gene - cells - localization - antibodies - oligosaccharides - glycoproteins
b1,4-Galactosylation of plant N-glycans is a prerequisite for commercial production of certain biopharmaceuticals in plants. Two different types of galactosylated N-glycans have initially been reported in plants as the result of expression of human b1,4-galactosyltransferase 1 (GalT). Here we show that these differences are associated with differences at its N-terminus: the natural short variant of human GalT results in hybrid type N-glycans, whereas the long form generates bi-antennary complex type N-glycans. Furthermore, expression of non-mammalian, chicken and zebrafish GalT homologues with N-termini resembling the short human GalT N-terminus also induce hybrid type N-glycans. Providing both non-mammalian GalTs with a 13 amino acid N-terminal extension that distinguishes the two naturally occurring forms of human GalT, acted to increase the levels of biantennary galactosylated N-glycans when expressed in tobacco leaves. Replacement of the cytosolic tail and transmembrane domain of chicken and zebrafish GalTs with the corresponding region of rat a2,6-sialyltransferase yielded a gene whose expression enhanced the level of bi-antennary galactosylation even further.
GtfA and GtfB are both required for protein O-glycosylation in Lactobacillus plantarum
Lee, I.C. ; Swam, I.I. van; Tomita, S. ; Morsomme, P. ; Rolain, T. ; Hols, P. ; Kleerebezem, M. ; Bron, P.A. - \ 2014
Journal of Bacteriology 196 (2014)9. - ISSN 0021-9193 - p. 1671 - 1682.
complete genome sequence - lactic-acid bacteria - escherichia-coli - campylobacter-jejuni - acidophilus ncfm - epithelial-cells - surface protein - rhamnosus gg - glycoproteins - binding
Acm2, the major autolysin of Lactobacillus plantarum WCFS1, was recently found to be O-glycosylated with N-acetylhexosamine, likely N-acetylglucosamine (GlcNAc). In this study, we set out to identify the glycosylation machinery by employing a comparative genomics approach to identify Gtf1 homologues, which are involved in fimbria-associated protein 1 (Fap1) glycosylation in Streptococcus parasanguinis. This in silico approach resulted in the identification of 6 candidate L. plantarum WCFS1 genes with significant homology to Gtf1, namely, tagE1 to tagE6. These candidate genes were targeted by systematic gene deletion, followed by assessment of the consequences on glycosylation of Acm2. We observed a changed mobility of Acm2 on SDS-PAGE in the tagE5E6 deletion strain, while deletion of other tagE genes resulted in Acm2 mobility comparable to that of the wild type. Subsequent mass spectrometry analysis of excised and in-gel-digested Acm2 confirmed the loss of glycosylation on Acm2 in the tagE5E6 deletion mutant, whereas a lectin blot using GlcNAc-specific succinylated wheat germ agglutinin (sWGA) revealed that besides Acm2, tagE5E6 deletion also abolished all but one other sWGA-reactive, protease-sensitive signal. Only complementation of both tagE5 and tagE6 restored those sWGA lectin signals, establishing that TagE5 and TagE6 are both required for the glycosylation of Acm2 as well as the vast majority of other sWGA-reactive proteins. Finally, sWGA lectin blotting experiments using a panel of 8 other L. plantarum strains revealed that protein glycosylation is a common feature in L. plantarum strains. With the establishment of these enzymes as protein glycosyltransferases, we propose to rename TagE5 and TagE6 as GtfA and GtfB, respectively.
Molecular characterization of the full-length L and M RNAs of Tomato yellow ring virus, a member of the genus Tospovirus
Chen, Tsung-Chi ; Li, Ju-Ting ; Fan, Ya-Shu ; Yeh, Yi-Chun ; Yeh, Shyi-Dong ; Kormelink, R.J.M. - \ 2013
Virus Genes 46 (2013)3. - ISSN 0920-8569 - p. 487 - 495.
spotted-wilt-virus - long-distance movement - nsm protein - nucleocapsid protein - plant-cells - sequence - glycoproteins - identification - polymerase - suppressor
Tomato yellow ring virus (TYRV), first isolated from tomato in Iran, was classified as a non-approved species of the genus Tospovirus based on the characterization of its genomic S RNA. In the current study, the complete sequences of the genomic L and M RNAs of TYRV were determined and analyzed. The L RNA has 8,877 nucleotides (nt) and codes in the viral complementary (vc) strand for the putative RNA-dependent RNA polymerase (RdRp) of 2,873 amino acids (aa) (331 kDa). The RdRp of TYRV shares the highest aa sequence identity (88.7 %) with that of Iris yellow spot virus (IYSV), and contains conserved motifs shared with those of the animal-infecting bunyaviruses. The M RNA contains 4,786 nt and codes in ambisense arrangement for the NSm protein of 308 aa (34.5 kDa) in viral sense, and the Gn/Gc glycoprotein precursor (GP) of 1,310 aa (128 kDa) in vc-sense. Phylogenetic analyses indicated that TYRV is closely clustered with IYSV and Polygonum ringspot virus (PolRSV). The NSm and GP of TYRV share the highest aa sequence identity with those of IYSV and PolRSV (89.9 and 80.2–86.5 %, respectively). Moreover, the GPs of TYRV, IYSV, and PolRSV share highly similar characteristics, among which an identical deduced N-terminal protease cleavage site that is distinct from all tospoviral GPs analyzed thus far. Taken together, the elucidation of the complete genome sequence and biological features of TYRV support a close ancestral relationship with IYSV and PolRSV.
Identification of alg3 in the mushroom-forming fungus Schizophyllum commune and analysis of the ¿alg3 knockout mutant
Berends, E. ; Lehle, L. ; Henquet, M. ; Hesselink, T. ; Wösten, H.A.B. ; Lugones, L.G. ; Bosch, H.J. - \ 2013
Glycobiology 23 (2013)2. - ISSN 0959-6658 - p. 147 - 154.
saccharomyces-cerevisiae - n-glycan - gene - glycoproteins - enzyme - mannosyltransferase - glucocerebrosidase - oligosaccharides - glycosylation - therapy
Alg3 of Saccharomyces cerevisiae catalyzes the mannosyl transfer from Man-P-Dol to Man(5)GlcNAc(2)-PP-Dol resulting in the formation of Man(6)GlcNAc(2)-PP-Dol, which is then further processed to the final precursor oligosaccharide Glc(3)Man(9)GlcNAc(2) for N-glycosylation of proteins. Here, we identified the alg3 gene of the mushroom-forming fungus Schizophyllum commune by homology search. Its function was confirmed by the complementation of the Delta alg3 strain of S. cerevisiae. Inactivation of alg3 in S. commune resulted in the production of predominantly Man(3)GlcNAc(2) protein-linked N-glycans. No impact on growth nor a developmental phenotype due to the deletion was observed. This provides a first step toward engineering of a homogeneous, humanized N-glycosylation pattern for the production of therapeutic glycoproteins in mushrooms.
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
Carp mucus and its role in mucosal defense
Marel, M.C. van der - \ 2012
Wageningen University. Promotor(en): Huub Savelkoul; D. Steinhagen, co-promotor(en): Jan Rombout. - S.l. : s.n. - ISBN 9789461734273 - 183
karper - cyprinus - slijm - verdedigingsmechanismen - vissen - immunologie - glycoproteïnen - carp - mucus - defence mechanisms - fishes - immunology - glycoproteins
Oligosaccharides in feces of breast- and formula-fed babies
Albrecht, S.A. ; Schols, H.A. ; Zoeren, D. van; Lingen, R.A. van; Groot Jebbink, L.J.M. ; Heuvel, E.G.H.M. van den; Voragen, A.G.J. ; Gruppen, H. - \ 2011
Carbohydrate Research : an international journal 346 (2011)14. - ISSN 0008-6215 - p. 2173 - 2181.
induced fluorescence detection - human-colon ecosystems - blood-group antigens - human-milk - capillary-electrophoresis - preterm infants - ce-lif - bacteria - glycoproteins - degradation
So far, little is known on the fate of oligosaccharides in the colon of breast- and formula-fed babies. Using capillary electrophoresis with laser induced fluorescence detector coupled to a mass spectrometer (CE–LIF–MSn), we studied the fecal oligosaccharide profiles of 27 two-month-old breast-, formula- and mixed-fed preterm babies. The interpretation of the complex oligosaccharide profiles was facilitated by beforehand clustering the CE–LIF data points by agglomerative hierarchical clustering (AHC). In the feces of breast-fed babies, characteristic human milk oligosaccharide (HMO) profiles, showing genetic fingerprints known for human milk of secretors and non-secretors, were recognized. Alternatively, advanced degradation and bioconversion of HMOs, resulting in an accumulation of acidic HMOs or HMO bioconversion products was observed. Independent of the prebiotic supplementation of the formula with galactooligosaccharides (GOS) at the level used, similar oligosaccharide profiles of low peak abundance were obtained for formula-fed babies. Feeding influences the presence of diet-related oligosaccharides in baby feces and gastrointestinal adaptation plays an important role herein. Four fecal oligosaccharides, characterized as HexNAc-Hex-Hex, Hex-[Fuc]-HexNAc-Hex, HexNAc-[Fuc]-Hex-Hex and HexNAc-[Fuc]-Hex-HexNAc-Hex-Hex, highlighted an active gastrointestinal metabolization of the feeding-related oligosaccharides. Their presence was linked to the gastrointestinal mucus layer and the blood-group determinant oligosaccharides therein, which are characteristic for the host’s genotype.
Partial Functional Rescue of Helicoverpa armigera Single Nucleocapsid Nucleopolyhedrovirus Infectivity by Replacement of F Protein with GP64 from Autographa californica Multicapsid Nucleopolyhedrovirus
Wang, M.L. ; Yin, F.F. ; Shen, S. ; Tan, Y. ; Deng, F. ; Vlak, J.M. ; Hu, Z.H. ; Wang, H.L. - \ 2010
Journal of Virology 84 (2010)21. - ISSN 0022-538X - p. 11505 - 11514.
envelope fusion protein - nuclear polyhedrosis-virus - baculovirus gp64 - lentiviral vectors - membrane-fusion - glycoproteins - cells - homolog - entry - ac23
Two distinct envelope fusion proteins (EFPs) (GP64 and F) have been identified in members of the Baculoviridae family of viruses. F proteins are found in group II nucleopolyhedroviruses (NPVs) of alphabaculoviruses and in beta- and deltabaculoviruses, while GP64 occurs only in group I NPVs of alphabaculoviruses. It was proposed that an ancestral baculovirus acquired the gp64 gene that conferred a selective advantage and allowed it to evolve into group I NPVs. The F protein is a functional analogue of GP64, as evidenced from the rescue of gp64-null Autographa californica multicapsid nucleopolyhedrovirus (MNPV) (AcMNPV) by F proteins from group II NPVs or from betabaculoviruses. However, GP64 failed to rescue an F-null Spodoptera exigua MNPV (SeMNPV) (group II NPV). Here, we report the successful generation of an infectious gp64-rescued group II NPV of Helicoverpa armigera (vHaBac Delta F-gp64). Viral growth curve assays and quantitative real-time PCR (Q-PCR), however, showed substantially decreased infectivity of vHaBac Delta F-gp64 compared to the HaF rescue control virus vHaBac Delta F-HaF. Electron microscopy further showed that most vHaBac Delta F-gp64 budded viruses (BV) in the cell culture supernatant lacked envelope components and contained morphologically aberrant nucleocapsids, suggesting the improper BV envelopment or budding of vHaBac Delta F-gp64. Bioassays using pseudotyped viruses with a reintroduced polyhedrin gene showed that GP64-pseudotyped Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus (HearNPV) significantly delayed the mortality of infected H. armigera larvae.
Analyses of human colonic mucus obtained by an in vivo sampling technique
Hamer, H.M. ; Jonkers, D.M.A.E. ; Loof, A. ; Vanhoutvin, S.A.L.W. ; Troost, F.J. ; Venema, K. ; Kodde, A. ; Koek, G.H. ; Schipper, R.G. ; Heerde, W.L. van; Brummer, R.J. - \ 2009
Digestive and Liver Disease 41 (2009)8. - ISSN 1590-8658 - p. 559 - 564.
inflammatory-bowel-disease - ulcerative-colitis - mucosal cytokine - trefoil peptide - sialic acids - filter-paper - mucins - rats - glycoproteins - resistance
Background The mucus layer is an important dynamic component of the epithelial barrier. It contains mucin glycoproteins and other compounds secreted by the intestinal epithelium, such as secretory IgA. However, a standardized in vivo sampling technique of mucus in humans is not yet available. Aim To assess the validity and feasibility of mucin and protein determinations in human colonic mucus collected under physiological conditions. Subjects and methods Triplicate colonic mucus samples were collected in 11 healthy volunteers using cytology brushes during sigmoidoscopy. As an indication of the quantity of collected mucus, total protein and mucin concentrations were determined by measuring oligosaccharide equivalents and monosaccharides. Also secretory IgA and sialic acid concentrations were determined and proteomic analysis was performed using surface enhanced laser desorption/ionization-time of flight-mass spectrometry. Results Mean values of secretory IgA and sialic acid corrected for the amount of mucus ranged from 0.16 to 1.81 g secretory IgA/mmol oligosaccharide equivalents and from 12.6 to 48.6 g sialic acid/mmol oligosaccharide equivalents. Proteomic analysis of mucus is feasible and cluster analysis showed subject specific profiles. Conclusion Using cytology brushes, human colonic mucus can be sampled and under physiological conditions. These samples could give information on the composition and quality of the mucus layer.
N-glycosylation in plants: science and application
Henquet, M.G.L. - \ 2009
Wageningen University. Promotor(en): Linus van der Plas; Dirk Bosch, co-promotor(en): Sander van der Krol. - [S.l.] : S.n. - ISBN 9789085852964 - 153
planten - plantenfysiologie - glycoproteïnen - enzymen - eiwitexpressieanalyse - plants - plant physiology - glycoproteins - enzymes - proteomics
In this thesis we set out to increase our knowledge of N-glycosylation in plants with a dual aim: (1) to develop tools for general and cell-specific glycoproteomics by which differences in the glycoproteome of plants in different specified cell types or under different conditions can be studied and (2) to increase our general understanding of N-glycosylation by analysis of the biosynthesis pathway and dissection of N-glycan function in plants.
(1) Identification of the glycoproteome from specific cell types during different physiological or developmental conditions provides valuable biological information. For the detection of glycoproteins with plant complex type glycans anti-HRP polyclonal serum is available. Using these antibodies differences were identified in the complex glycoproteome of leaf epidermal and mesophyll cells and it was demonstrated that these differences vary in other plant tissues. Cell specific tagging with complex glycans was accomplished by cell specific complementation of the cgl mutant, demonstrating the power of this technique to detect subtle differences within a tissue that are lost in whole tissue protein extracts of wild-type plants. With anti-HRP we were not able to purify and validate the complex glycoproteome as predicted by bioinformatics. For glycoproteomics, the availability of probes that interact with specific plant N-glycans would be very helpful for the isolation and subsequent analysis of specific sub-pools of glycoproteins. In addition, such probes can aid the analysis of glycan modification on target proteins. For this reason, we used two alternative approaches to isolate monoclonal antibodies that can recognise plant complex N-glycans, neither of which yielded a probe with the desired properties. Suggestions are given on how strategies to select specific plant N-glycan antibodies may be improved.
(2) N-glycan synthesis and processing is performed by sequential activity of enzymes in the ER and Golgi. Two of these enzymes were studied in more detail in this thesis: ALG3, involved in lipid-linked glycan synthesis in the ER, and GnTI involved in Golgi localized N-glycan processing to complex glycans. The ALG3 gene of Arabidopsis was identified and characterization of an alg3 mutant provided information on the in vivo substrate characteristics of various downstream enzymes, including the OST complex, and of consequences for the Unfolded Protein Response (UPR) in plants. Analysis indicated that ER resident glycoproteins from this mutant have predominantly Man3-5GlcNAc2 N-glycans. For this reason the alg3-2 mutant was used to produce an ER retained variant of monoclonal antibodies in seeds.
It was demonstrated that human GnTI is much less efficient in complementing the cgl (GnTI) mutant in Arabidopsis than the homologous Arabidopsis gene. Analysis showed that this was not due to RNA expression but to differences in protein stability and reduced catalytic activity of Human GnTI. The results also suggest some form of competition between human and plant GnTI when produced in the same cell, leading to partially mutual exclusive targeting to presumed Golgi stacks.
High-throughput screening of monoclonal antibodies against plant cell wall glycans by hierarchical clustering of their carbohydrate microarray binding profiles
Moller, I. ; Marcus, S.E. ; Haeger, A. ; Verhertbruggen, Y. ; Verhoef, R.P. ; Schols, H.A. ; Ulvskov, P. ; Mikkelsen, J.D. ; Knox, J.P. ; Willats, W.G.T. - \ 2008
Glycoconjugate Journal 25 (2008)1. - ISSN 0282-0080 - p. 37 - 48.
oligosaccharide microarrays - arabinogalactan-proteins - glycomics - pectin - polysaccharides - generation - epitope - carrot - homogalacturonan - glycoproteins
Antibody-producing hybridoma cell lines were created following immunisation with a crude extract of cell wall polymers from the plant Arabidopsis thaliana. In order to rapidly screen the specificities of individual monoclonal antibodies (mAbs), their binding to microarrays containing 50 cell wall glycans immobilized on nitrocellulose was assessed. Hierarchical clustering of microarray binding profiles from newly produced mAbs, together with the profiles for mAbs with previously defined specificities allowed the rapid assignments of mAb binding to antigen classes. mAb specificities were further investigated using subsequent immunochemical and biochemical analyses and two novel mAbs are described in detail. mAb LM13 binds to an arabinanase-sensitive pectic epitope and mAb LM14, binds to an epitope occurring on arabinogalactan-proteins. Both mAbs display novel patterns of recognition of cell walls in plant materials. Keywords Carbohydrate microarrays - Plant cell walls - Monoclonal antibodies - Hierarchical clustering
Deglycosylation of ovalbumin prohibits formation of a heat-stable conformer
Groot, J. de; Kosters, H.A. ; Jongh, H.H.J. de - \ 2007
Biotechnology and Bioengineering 97 (2007)4. - ISSN 0006-3592 - p. 735 - 741.
monoclonal-antibodies - structural-properties - s-ovalbumin - carbohydrate - protein - transformation - glycoproteins - resolution - stability - sequence
To study the influence of the carbohydrate-moiety of ovalbumin on the formation of the heat-stable conformer S-ovalbumin, ovalbumin is deglycosylated with PNGase-F under native conditions. Although the enzymatic deglycosylation procedure resulted in a complete loss of the ability to bind to Concavalin A column-material, only in about 50% the proteins lost their complete carbohydrate moiety, as demonstrated by mass spectrometry and size exclusion chromatography. Thermal stability and conformational changes were determined using circular dichroism and differential scanning calorimetry and demonstrated at ambient temperature no conformational changes due to the deglycosylation. Also the denaturation temperature of the processed proteins remained the same (77.4 ± 0.4°C). After heat treatment of the processed protein at 55°C and pH 9.9 for 72 h, the condition that converts native ovalbumin into the heat-stable conformer (S-ovalbumin), only the material with the intact carbohydrate moiety forms this heat-stable conformer. The material that effectively lost its carbohydrate moiety appeared fully denatured and aggregated due to these processing conditions. These results indicate that the PNGase-F treatment of ovalbumin prohibits the formation and stabilization of the heat-stable conformer S-ovalbumin. Since S-ovalbumin in egg protein samples is known to affect functional properties, this work illustrates a potential route to control the quality of egg protein ingredients
Tomato spotted wilt virus Gc and N proteins interact in vivo
Snippe, M. ; Borst, J.W. ; Goldbach, R.W. ; Kormelink, R.J.M. - \ 2007
Virology 357 (2007)2. - ISSN 0042-6822 - p. 115 - 123.
nucleocapsid protein - fluorescence microscopy - mammalian-cells - matrix protein - living cells - fever virus - membrane - glycoproteins - localization - microtubules
Tomato spotted wilt virus (TSWV) virions consist of a nucleocapsid core surrounded by a membrane containing glycoproteins Gn and Gc. To unravel the protein interactions involved in the membrane acquisition of RNPs, TSWV nucleocapsid protein (N), Gn and Gc were expressed and analyzed in BHK21 cells. Upon coexpression of Gn, Gc and N, a partial colocalization of N with both glycoproteins was observed in the Golgi region. In contrast, upon coexpression of Gc and N in the absence of Gn, both proteins colocalized to a distinct non-Golgi perinuclear region. Using FLIM and FRET, interaction was demonstrated between N and Gc, but not between N and Gn, and was only observed in the region where both proteins accumulated. The genuine character of N¿Gc interaction was confirmed by its presence in purified virus and RNP preparations. The results are discussed in view of TSWV particle assembly taking place at the Golgi complex
Electrospray ionization quadrupole time-of-flight tandem mass spectrometric analysis of hexamethylenediamine-modified maltodextrin and dextran
Sisu, E. ; Bosker, W.T.E. ; Norde, W. ; Slaghek, T.M. ; Timmermans, J.W. ; Peter-Katalinic, J. ; Cohen Stuart, M.A. ; Zamfir, A.D. - \ 2006
Rapid Communications in Mass Spectrometry 20 (2006)2. - ISSN 0951-4198 - p. 209 - 218.
capillary-zone-electrophoresis - oligosaccharides - derivatives - chromatography - glycoproteins - copolymers - resolution
A combined methodology for obtaining at the preparative scale and characterization by nanoelectrospray ionization (nanoESI) quadrupole time-of-flight (QTOF) mass spectrometry (MS) and tandem MS (MS/MS) of linear polysaccharides modified at the reducing end is presented. Two polydisperse maltodextrins (1000 and 3000 Da) and a high molecular weight polydisperse dextran (6000 Da) were coupled with hexamethylenediamine (HMD). The coupling products were analyzed by nanoESI-QTOF-MS in the positive ion mode and MS/MS using collision-induced dissociation (CID) at low energies. In the HMD-M1000 mixture, the polysaccharide chains containing from 2 to 8 Glc residues were detected, while in HMD-M3000 we identified a complete series of chains containing from 8 to 21 Glc moieties. The employed ESI conditions enhanced the detection of chains with up to 46 Glc residues in the HMD-D6000 sample. By optimized MS/MS, HMD-modified polysaccharides of 3,4, 5, 12 and 46 degrees of polymerization yielded product ion spectra exhibiting the whole set of Y- and B-fragment ions. The MS structural data were obtained within a few minutes of signal acquisition, with a sample consumption situating the analysis sensitivity in the picomolar range
Tomato spotted wilt virus particle assembly : studying the role of the structural proteins in vivo
Snippe, M. - \ 2006
Wageningen University. Promotor(en): R.W. Goldbach, co-promotor(en): Richard Kormelink. - [S.l. ] : S.n. - ISBN 9085043263 - 128
solanum lycopersicum - tomaten - tomatenbronsvlekkenvirus - tospovirus - viruseiwitten - virale regulatoire eiwitten - glycoproteïnen - fluorescentiemicroscopie - genexpressieanalyse - tomatoes - tomato spotted wilt virus - viral proteins - viral regulatory proteins - glycoproteins - fluorescence microscopy - genomics
Function, oligomerization and N-linked glycosylation of the Helicoverpa armigera single nucleopolyhedrovirus envelope fusion protein
Long, G. ; Westenberg, M. ; Wang, H. ; Vlak, J.M. ; Hu, Z. - \ 2006
Journal of General Virology 87 (2006)4. - ISSN 0022-1317 - p. 839 - 846.
californica multicapsid nucleopolyhedrovirus - nuclear polyhedrosis-virus - viral membrane-fusion - baculovirus gp64 - cell-lines - cross-linking - insect - genome - glycoproteins - activation
In the family Baculoviridae, two distinct envelope fusion proteins are identified in budded virions (BVs). GP64 is the major envelope fusion protein of group I nucleopolyhedrovirus (NPV) BVs. An unrelated type of envelope fusion protein, named F, is encoded by group II NPVs. The genome of Helicoverpa armigera (Hear) NPV, a group II NPV of the single nucleocapsid or S type, also encodes an F-like protein: open reading frame 133 (Ha133). It was demonstrated by N-terminal sequencing of the major 59 kDa protein present in HearNPV BV that this protein is one of the two F subunits: F1 (transmembrane subunit of 59 kDa) and F2 (surface subunit of 20 kDa), both the result of cleavage by a proprotein convertase and disulfide-linked. The HearNPV F protein proved to be a functional analogue of GP64, as the infectivity of an AcMNPV gp64-deletion mutant was rescued by the introduction of the HearNPV F gene. It was also demonstrated by chemical cross-linking that HearNPV F is present in BVs as an oligomer whereby, unlike GP64, disulfide bonds are not involved. Deglycosylation assays indicated that both F1 and F2 possess N-linked glycans. However, when F was made in Hz2E5 cells, these glycans did not have an ¿-1-3 core fucose modification that usually occurs in insect cells. As ¿-1-3 core fucose is a major inducer of an allergic response in humans, the present observation makes the HearNPV-Hz2E5 system an attractive alternative for the production of recombinant glycoproteins for therapeutic use in humans
Virulence of Newcastle disease virus is determined by the cleavage site of the fusion protein and by both the stem region and globular head of the haemagglutinin-neuraminidase protein
Leeuw, O.S. de; Koch, G. ; Hartog, L. ; Ravenshorst, N. ; Peeters, B.P.H. - \ 2005
Journal of General Virology 86 (2005)6. - ISSN 0022-1317 - p. 1759 - 1769.
virus v-protein - interferon-antagonist - nucleotide-sequence - intergenic regions - f-protein - pathogenicity - glycoproteins - pathogenesis - expression - generation
Virulence of Newcastle disease virus (NDV) is mainly determined by the amino acid sequence surrounding the fusion (F) protein cleavage site, since host proteases that cleave the F protein of virulent strains are present in more tissues than those that cleave the F protein of non-virulent strains. Nevertheless, comparison of NDV strains that carry exactly the same F protein cleavage site shows that significant differences in virulence still exist. For instance, virulent field strain Herts/33 with the F cleavage site 112RRQRRF117 had an intracerebral pathogenicity index of 1.88 compared with 1.28 for strain NDFLtag, which has the same cleavage site. This implies that additional factors contribute to virulence. After generating an infectious clone of Herts/33 (FL-Herts), we were able to map the location of additional virulence factors by exchanging sequences between FL-Herts and NDFLtag. The results showed that, in addition to the F protein cleavage site, the haemagglutinin-neuraminidase (HN) protein also contributed to virulence. The effect of the HN protein on virulence was most prominent after intravenous inoculation. Interestingly, both the stem region and the globular head of the HN protein seem to be involved in determining virulence