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.
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
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.
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
Role of the envelope glycoproteins in the infection cycle of tomato spotted wilt virus
Kikkert, M. - \ 1999
Agricultural University. Promotor(en): R.W. Goldbach; R. Kormelink. - S.l. : Kikkert - ISBN 9789058080585 - 117
tomatenbronsvlekkenvirus - plantenvirussen - plantenziekteverwekkers - plantenziekten - infectie - glycoproteïnen - tomato spotted wilt virus - plant viruses - plant pathogens - plant diseases - infection - glycoproteins
<p><em>Tomato spotted wilt virus</em> (TSWV) forms the type member of the genus <em>Tospovirus</em> , which today harbors more than twelve different species. TSWV is able to infect an enormous variety of different plants, to which it often causes devastating effects, resulting in severe economical losses. Among the plant viruses, TSWV and the other tospoviruses form a distinct group. Taxonomically, they surprisingly do not belong to a plant virus family, but to a virus family which further consists of animal-infecting viruses, the <em>Bunyaviridae</em> . Consequently, they harbor features that are more common to animal-infecting viruses than to plant viruses. The most eye-catching animal-infecting virus-like feature of the tospoviruses is their envelope, in which two viral surface glycoproteins are embedded, denoted G1 and G2. These surface glycoproteins are designed for interaction with receptors, an important step in the infection of animals, but useless in the infection of plants. The plant-infecting tospoviruses are transmitted by thrips, in which they also replicate, and for the entry and circulation of the virus through this insect the glycoproteins are essential. This ensures their continues presence despite their lack of function during the infection of plants. The structure and function of the TSWV glycoproteins during infection in plants and insects form the subject of this thesis, of which the contents will be summarized in the next paragraphs, and also visually represented in Fig. 1.</p><p><em>First develop the tools...</em></p><p>Although the TSWV glycoproteins do not have a crucial function in the plant, they do play an essential role in the formation of virus particles. At the onset of these studies, however, no clear view of this morphogenesis process was available, so it was the obvious first goal to unravel this process for TSWV in plants. In the past, virus associated structures observed during the infection of whole plants were reported, but thirty five years of observations had not resulted in a clear model of the particle morphogenesis. Useful antibodies against the separate TSWV proteins had not been available, and most importantly, a system in which a synchronous infection could be investigated lacked, so that interpretation of the chrology of the events in the morphogenesis had been difficult. <em>Chapter 2</em> of this thesis describes the development of a protoplast infection system for TSWV, which enabled the study of a synchronous TSWV infection in plant cells. Using newly produced antibodies against the viral glycoproteins together with antibodies against the nucleoprotein, it could be shown that a full, synchronous, TSWV infection is achieved, by the PEG-mediated inoculation of freshly isolated <em>Nicotiana rustica</em> protoplasts with freshly (and quickly) isolated TSWV particles. Similar inoculation of <em>Vigna unguiculata</em> protoplasts did not result in a full infection, since the production of enveloped particles was hampered due to low expression of the viral glycoproteins.</p><p><em>...to reveal the essence of TSWV particle making...</em></p>Using the system, based on <em>N. rustica</em> protoplasts, the different virus associated structures could be assigned a chronological position in the morphogenesis process. This view of the chronology of the process revealed that the so-called paired parrallel membranes and doubly enveloped particles form essential intermediates in the process, which precede the accumulation of singly enveloped particles in the lumen of ER membranes. Using specific antibodies against the TSWV structural proteins, as well as antibodies against plant cell organels, the model could be completed ( <em>Chapter 3</em> ). TSWV structural components, nucleocapsids as well as glycoproteins, accumulate at Golgi membranes, which are consequently modified to form the paired parrallel membranes. Doubly enveloped particles are formed by the so-called "wrapping" of these viral glycoprotein containing membranes around nucleocapsid cores, a process unique among plant viruses. The subsequent step is the fusion of these doubly enveloped particles with each other and specifically with ER membranes. This results in the formation of singly enveloped particles that accumulate within the ER.</p><p><em>...and then look what causes all this...</em></p><p>After the complete model of the morphogenesis became available, the next step was to investigate what (molecular) features of especially the glycoproteins regulate the process. An important observation in the morphogenesis process is the apparent accumulation of viral glycoproteins in the Golgi system. This may, analogous to other enveloped viruses, be caused by the specific targeting of the glycoproteins to this organel due to a retention signal. The trafficking and retention behavior of TSWV glycoproteins was investigated in mammalian cells, the results of which are described in <em>Chapter 4</em> . TSWV G1 and G2 accumulate in the Golgi system when expression together, which indeed implicates that at least one of the proteins must harbor a Golgi retention signal. Separate expression of G1 and G2 revealed that the retention signal is present in G2. G1 on its own is transport incompetent, but this can be rescued by the co-expression with G2, which suggests that G1 is dependent on, and interacts with G2 during transport and retention. These molecular features, identified in mammalian cells, are most probably also functional in plant cells, causing the observed accumulation of glycoproteins in the plant Golgi system during infection. The TSWV glycoproteins thereby show their crucial role in directing the particle morphogenesis process.</p><p><em>...and how these particles interact with thrips.</em></p><p>Once the particles are formed and accumulated inside ER membranes, they await the uptake by the thrips vector to be transferred to another plant. Earlier research has shown that between this uptake and the release of virus there is replication and circulation of TSWV in the thrips. However, nothing was known about the molecular interactions between TSWV proteins and proteins of the thrips during this process. In <em>Chapter 5</em> an overlay blot technique was used to investigate the possible binding of TSWV structural proteins with thrips proteins, that could be potential receptors involved in entry or circulation of TSWV. A 94 kDa thrips protein was identified, displaying specific binding to TSWV G2 protein. This 94 kDa protein was found in known vectors of TSWV, and also in a non-vector thrips species, albeit it not in the larval stages of the latter. Although a receptor is anticipated in the gut of the vectoring insects, the 94 kDa protein is not found there. It is however present in all other parts of the thrips body, suggesting that it may have a role during the replication and circulation of the virus.</p><CENTER><p><strong>Fig. 1</strong><br/><img src="/wda/abstracts/ab2637.gif" WIDTH="470" HEIGHT="358" ALT="Fig. 1" BORDER="0"/><br/><em>Diagram of TSWV infection in a plant cell, indicating the scope of the experimental chapters of this thesis</em></CENTER>
Bepaling van glycomacropeptide (GMP) in melkpoeders m.b.v. NIR
Boers, E.A.M. ; Frankhuizen, R. ; Veen, N.G. van der - \ 1984
Wageningen : RIKILT (Rapport / RIKILT 84.88)
melkpoeder - glycoproteïnen - infraroodspectrofotometrie - wei-eiwit - caseïne - dried milk - glycoproteins - infrared spectrophotometry - whey protein - casein
Nagegaan is of magere melkpoeders met behulp van NIR gescreend kunnen worden op het gehalte aan glycomacropeptide.