Staff Publications

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.

    Full text documents are added when available. The database is updated daily and currently holds about 240,000 items, of which 72,000 in open access.

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The positively-selected effector MiL648 of Meloidogyne incognita contributes to nematode virulence through its interactions with the 12-oxophytodienoate reductase OPR2 in tomato
Verhoeven, Ava ; Tacken, Wannes ; Wesselink, Thomas ; Finkers-Tomczak, A.M. ; Prins, J.C.P. ; Raaij, D.R. van; Varossieau, K. ; Overmars, H.A. ; Slootweg, E.J. ; Goverse, A. ; Smant, G. - \ 2019
Range-expansion effects on the belowground plant microbiome
Ramirez, Kelly S. ; Snoek, L.B. ; Koorem, Kadri ; Geisen, Stefan ; Bloem, L.J. ; Hooven, Freddy ten; Kostenko, Olga ; Krigas, Nikos ; Manrubia, Marta ; Caković, Danka ; Raaij, Debbie van; Tsiafouli, Maria A. ; Vreš, Branko ; Čelik, Tatjana ; Weser, Carolin ; Wilschut, Rutger A. ; Putten, Wim H. van der - \ 2019
Nature Ecology & Evolution 3 (2019)4. - ISSN 2397-334X - p. 604 - 611.

Plant range expansion is occurring at a rapid pace, largely in response to human-induced climate warming. Although the movement of plants along latitudinal and altitudinal gradients is well-documented, effects on belowground microbial communities remain largely unknown. Furthermore, for range expansion, not all plant species are equal: in a new range, the relatedness between range-expanding plant species and native flora can influence plant–microorganism interactions. Here we use a latitudinal gradient spanning 3,000 km across Europe to examine bacterial and fungal communities in the rhizosphere and surrounding soils of range-expanding plant species. We selected range-expanding plants with and without congeneric native species in the new range and, as a control, the congeneric native species, totalling 382 plant individuals collected across Europe. In general, the status of a plant as a range-expanding plant was a weak predictor of the composition of bacterial and fungal communities. However, microbial communities of range-expanding plant species became more similar to each other further from their original range. Range-expanding plants that were unrelated to the native community also experienced a decrease in the ratio of plant pathogens to symbionts, giving weak support to the enemy release hypothesis. Even at a continental scale, the effects of plant range expansion on the belowground microbiome are detectable, although changes to specific taxa remain difficult to decipher.

The nematode effector MiL648 contributes to virulence through its interactions with the 12-oxophytodienoate reductase OPR2 in tomato
Verhoeven, Ava ; Yang, Yixuan ; Raaij, D.R. van; Steenbrugge, Joris van; Goverse, A. ; Smant, G. - \ 2019
The positively-selected effector MiL648 of the root-knot nematode Meloidogyne incognita plays a role in tomato infections
Verhoeven, Ava ; Put, Sanne ; Slootweg, E.J. ; Raaij, D.R. van; Overmars, H.A. ; Goverse, A. ; Smant, G. - \ 2018
Insight in the molecular basis of virulence can help us to identify new and more durable sources of resistance to the root-knot nematode Meloidogyne incognita in tomato. Recently, we identified the effector MiL648 by applying a genome-wide scan for positively selected secreted proteins. To acquire a better understanding of the function of MiL648, we are currently functionally characterising the protein in plants using host-induced gene silencing. Several in planta targets of the putative effector have now been found by a yeast-two-hybrid screening and interactions were checked using Co-IPs. To identify the role of the host targets in the infection process, homologs of the host targets were knocked out in Arabidopsis.
Functional characterisation of the positively-selected effector MiL648 of the root-knot nematode Meloidogyne incognita
Verhoeven, Ava ; Prins, J.C.P. ; Finkers-Tomczak, A.M. ; Slootweg, E.J. ; Varossieau, K. ; Raaij, D.R. van; Goverse, A. ; Smant, G. - \ 2018
Granulocyte-macrophage colony-stimulating factor negatively regulates early IL-10-mediated responses
Wilbers, Ruud H.P. ; Westerhof, Lotte B. ; Raaij, Debbie R. Van; Bakker, Jaap ; Smant, Geert ; Schots, Arjen - \ 2018
Future Science OA 14 (2018)4. - ISSN 2056-5623
Aim: Treatment of inflammatory disorders relies on the intervention in immune responses thereby restoring homeostasis. IL-10 is a cytokine with therapeutic potential, but until now has not been as successful as previously anticipated. A reason for this may be that IL-10 responsiveness depends on the environment of the inflamed tissue. In this study we investigated whether GM-CSF is able to influence IL-10-mediated responses. Methodology: Dendritic cells and macrophages were differentiated from mouse bone marrow and treated or depleted from GM-CSF prior to analyze their response to IL-10. Activity was assessed by measuring cytokine expression upon lipopolysaccharide stimulation, IL-10-induced signaling and downstream gene expression. Conclusion: This study describes that GM-CSF negatively regulates IL-10-mediated responses.
Identification of differences in health impact modelling of salt reduction
Hendriksen, Marieke A.H. ; Geleijnse, Johanna M. ; Raaij, Joop M.A. Van; Cappuccio, Francesco P. ; Cobiac, Linda C. ; Scarborough, Peter ; Nusselder, Wilma J. ; Jaccard, Abbygail ; Boshuizen, Hendriek C. - \ 2017
PLoS ONE 12 (2017)11. - ISSN 1932-6203
We examined whether specific input data and assumptions explain outcome differences in otherwise comparable health impact assessment models. Seven population health models estimating the impact of salt reduction on morbidity and mortality in western populations were compared on four sets of key features, their underlying assumptions and input data. Next, assumptions and input data were varied one by one in a default approach (the DYNAMO-HIA model) to examine how it influences the estimated health impact. Major differences in outcome were related to the size and shape of the dose-response relation between salt and blood pressure and blood pressure and disease. Modifying the effect sizes in the salt to health association resulted in the largest change in health impact estimates (33% lower), whereas other changes had less influence. Differences in health impact assessment model structure and input data may affect the health impact estimate. Therefore, clearly defined assumptions and transparent reporting for different models is crucial. However, the estimated impact of salt reduction was substantial in all of the models used, emphasizing the need for public health actions.
Re-evaluation of IL-10 signaling reveals novel insights on the contribution of the intracellular domain of the IL-10R2 chain
Wilbers, Ruud H.P. ; Raaij, Debbie R. Van; Westerhof, Lotte B. ; Bakker, Jaap ; Smant, Geert ; Schots, Arjen - \ 2017
PLoS ONE 12 (2017)10. - ISSN 1932-6203
Interleukin-10 (IL-10) is an anti-inflammatory cytokine that plays a key role in maintainingimmune homeostasis. IL-10-mediated responses are triggered upon binding to a heterodimericreceptor complex consisting of IL-10 receptor (IL-10R)1 and IL-10R2. Engagementof the IL-10R complex activates the intracellular kinases Jak1 and Tyk2, but the exact rolesof IL-10R2 and IL-10R2-associated signaling via Tyk2 remain unclear. To elucidate the contributionof IL-10R2 and its signaling to IL-10 activity, we re-evaluated IL-10-mediatedresponses on bone marrow-derived dendritic cells, macrophages and mast cells. By usingbone marrow from IL-10R-/- mice it was revealed that IL-10-mediated responses depend onboth IL-10R1 and IL-10R2 in all three cell types. On the contrary, bone marrow-derived cellsfrom Tyk2-/- mice showed similar responses to IL-10 as wild-type cells, indicating that signalingvia this IL-10R2-associated kinase only plays a limited role. Tyk2 was shown to controlthe amplitude of STAT3 activation and the up-regulation of downstream SOCS3 expression.SOCS3 up-regulation was found to be cell-type dependent and correlated with the lack ofearly suppression of LPS-induced TNF-α in dendritic cells. Further investigation of the IL-10R complex revealed that both the extracellular and intracellular domains of IL-10R2 influencethe conformation of IL-10R1 and that both domains were required for transducing IL-10 signals. This observation highlights a novel role for the intracellular domain of IL-10R2 inthe molecular mechanisms of IL-10R activation.
Functional characterization of the positively-selected effector MiL648 of the root-knot nematode Meloidogyne incognita
Verhoeven, Ava ; Prins, J.C.P. ; Finkers-Tomczak, A.M. ; Slootweg, E.J. ; Varossieau, K. ; Raaij, D.R. van; Pomp, H. ; Goverse, A. ; Smant, G. - \ 2017
Physical Interaction of T Cells with Dendritic Cells Is Not Required for the Immunomodulatory Effects of the Edible Mushroom Agaricus subrufescens
Wilbers, Ruud H.P. ; Westerhof, Lotte B. ; Velde, Jan van de; Smant, Geert ; Valkenburg-van Raaij, Debbie ; Sonnenberg, Anton S.M. ; Bakker, Jaap ; Schots, Arjen - \ 2016
Frontiers in Immunology 7 (2016). - ISSN 1664-3224
Mushrooms are well known for their immunomodulating capacities. However, little is known about how mushroom-stimulated dendritic cells (DCs) affect T cells. Therefore, we investigated the effect of mushroom compounds derived from seven edible mushroom species on DCs, their fate in DCs, and the effect of the mushroom-stimulated DCs on T cells. Each mushroom species stimulated DCs in a different manner as was revealed from the DC’s cytokine response. Assessing DC maturation revealed that only one mushroom species, Agaricus subrufescens, induced complete DC maturation.
The other six mushroom species upregulated MHC-II and CD86 expression, but did not significantly affect the expression of CD40 and CD11c. Nevertheless, mushroom compounds of all investigated mushroom species are endocytosed by DCs. Endocytosis is most likely mediated by C-type lectin receptors (CLRs) because CLR binding is Ca2+ dependent, and EGTA reduces TNF-α secretion with more than 90%. Laminarin partly inhibited TNF-α secretion indicating that the CLR dectin-1, among other CLRs, is involved in binding mushroom compounds. Stimulated DCs were shown to stimulate T cells; however, physical contact of DCs and T cells is not required. Because CLRs seem to play a prominent role in DC stimulation, mushrooms may function as a carbohydrate
containing adjuvant to be used in conjunction with anti-fungal vaccines.
Are positively-selected effectors of the root-knot nematode Meloidogyne incognita involved in plant resistance
Verhoeven, Ava ; Prins, J.C.P. ; Finkers-Tomczak, A.M. ; Varossieau, K. ; Raaij, D.R. van; Pomp, H. ; Goverse, A. ; Smant, G. - \ 2016

The tropical root-knot nematode Meloidogyne incognita is a global problem in tomato and many other vegetable crops. The obligate endoparasitic M. incognita can cause about 5% crop loss annually. Chemical pesticides can no longer be used to control M. incognita because of environmental and public health concerns. Meanwhile, the most frequently used Mi-1 resistance in tomato is increasingly being broken by virulent populations of M. incognita. Virulent root knot nematodes are thought to have acquired effectors that enable them to avoid recognition by resistance proteins in plants. Therefore, insight into the repertoire of effectors of M. incognita can help to identify new sources of resistance to which the field populations of nematodes in tomato-producing areas have not yet adapted.

To identify positively-selected effector families in the genome sequence of M. incognita, a bioinformatics pipeline centred on the algorithms PRANK, Codeml, and Phobius was constructed. In total, 77 positively-selected clusters of paralogous genes were identified in the genome of M. incognita, which encode a total of 254 secretory proteins. The effectiveness of the pipeline was demonstrated by the fact that several positively-selected clusters of paralogous genes have been identified as effectors of M. incognita in earlier studies. Highly ranking candidate effectors from the pipeline are currently being functionally characterized in plants using transient and stable overexpression and using host-delivered RNA-interference in nematodes.

Codon use and mRNA structure analyses across kingdoms indicates selection on both mRNA stability and translatability
Westerhof, Lotte - \ 2016
Codon use and mRNA structure analyses across kingdoms indicates selection on both mRNA stability and translatability Lotte B. Westerhof, Mark G. Sterken, Ruud H.P. Wilbers, Lucia Luijben, Debbie R. van Raaij, L. Basten Snoek, Jaap Bakker and Arjen Schots To boost heterologous protein production the codon use of a gene of interest is often adapted to reflect the expression host’s codon use in highly expressed genes (optimal codons). However, the results obtained with this strategy are variable. A comparison between the overall codon use and the codon use in highly expressed genes of several plant species revealed that optimal codons are not always the codons of which the use is most increased with expression. Although the codon composition of highly expressed genes differs between monocots and dicots, often the same codons are linked strongest to expression (here after named expression codons). We used these conserved expression codons to optimise the codon composition of three genes, which enhanced protein yield significantly upon stable and transient expression in plants. Upon stable transformation both transcript levels and protein yield per transcript increased. Next, we analysed whether this expression-linked codon bias found in plants also extends to other kingdoms of life. Thereto, expression-linked codon use was investigated in Escherichia coli (Bacteria), Saccharomyces cerevisiae (Fungi), Caenorhabditis elegans (Animalia) and Mus musculus (Animalia) using more than 250 microarrays per species. We found that a common codon use bias exits over all species. In addition, computational analyses of various mRNA characteristics revealed a similar selection pressure across kingdoms that increases both stability and translatability. Combining gene expression data with available protein abundance data showed that an increased number of stem-loop transitions together with a reduction of stem size increases translation efficiency. An algorithm was developed that combines the use of optimal and expression codons to create an ideal mRNA structure for any given gene. This algorithm was again tested in plants and lead to a significant increase in protein production. The spin off company TripleT Biosciences combines this algorithm with all available knowledge on codon use and offers a codon optimization tool.
Engineering of plants for the expression of helminth glycoproteins with their native N-glycan structures
Wilbers, Ruud - \ 2016
Engineering plants for the expression of helminth glycoproteins with their native N-glycan structures Ruud H.P. Wilbers1, Lotte B. Westerhof 1, Bart Everts2, Kim van Noort 1, Debbie R. van Raaij1, Dieu-Linh Nguyen2, Maria Yazdanbakhsh2, Cornelis H. Hokke2 and Arjen Schots1 1 Laboratory of Nematology, Wageningen University, Wageningen, The Netherlands 2 Department of Parasitology, Leiden University Medical Center, Leiden, The Netherlands Schistosoma mansoni is a parasitic trematode that, like other helminths, secretes immunomodulatory proteins. These secreted proteins are main topics of research as they are possible vaccine candidates or may have therapeutic potential to treat inflammatory disorders. Many helminth secretory proteins carry complex N-glycans, but the exact role of these N-glycans on immunomodulatory properties remains to be elucidated. As the purification of a single glycoprotein from S. mansoni is inefficient and unsustainable, a platform is required that enables production of such glycoproteins. Here we show that S. mansoni-derived glycoproteins can be efficiently produced in plants. Furthermore, we have engineered the plant glycosylation machinery to synthesise N-glycans carrying structures like Lewis X or LDNF. Altogether, our results demonstrate that plants are an excellent platform for the expression of helminth glycoproteins with their native N-glycans. This opens up a new field of research and might lead to the identification of novel therapeutic targets.
Engineering of plants for the expression of helminth glycoproteins with their native N-glycan structures
Wilbers, R.H.P. ; Westerhof, L.B. ; Noort, Kim van; Nguyen, D.L. ; Smant, G. ; Bakker, J. ; Hokke, C.H. ; Schots, A. - \ 2016
- 1 p.
Engineering plants for the expression of helminth glycoproteins with their native N-glycan structures


Ruud H.P. Wilbers1, Lotte B. Westerhof 1, Bart Everts2, Kim van Noort 1, Debbie R. van Raaij1, Dieu-Linh Nguyen2, Maria Yazdanbakhsh2, Cornelis H. Hokke2 and Arjen Schots1

1 Laboratory of Nematology, Wageningen University, Wageningen, The Netherlands
2 Department of Parasitology, Leiden University Medical Center, Leiden, The Netherlands


Schistosoma mansoni is a parasitic trematode that, like other helminths, secretes immunomodulatory proteins. These secreted proteins are main topics of research as they are possible vaccine candidates or may have therapeutic potential to treat inflammatory disorders. Many helminth secretory proteins carry complex N-glycans, but the exact role of these N-glycans on immunomodulatory properties remains to be elucidated. As the purification of a single glycoprotein from S. mansoni is inefficient and unsustainable, a platform is required that enables production of such glycoproteins. Here we show that S. mansoni-derived glycoproteins can be efficiently produced in plants. Furthermore, we have engineered the plant glycosylation machinery to synthesise N-glycans carrying structures like Lewis X or LDNF. Altogether, our results demonstrate that plants are an excellent platform for the expression of helminth glycoproteins with their native N-glycans. This opens up a new field of research and might lead to the identification of novel therapeutic targets.
Codon use and mRNA structure analyses across kingdoms indicates selection on both mRNA stability and translatability
Westerhof, L.B. ; Sterken, M.G. ; Wilbers, R.H.P. ; Luijben, Lucia ; Raaij, D.R. van; Snoek, L.B. ; Bakker, J. ; Schots, A. - \ 2016
- 1 p.
Codon use and mRNA structure analyses across kingdoms indicates selection on both mRNA stability and translatability

Lotte B. Westerhof, Mark G. Sterken, Ruud H.P. Wilbers, Lucia Luijben, Debbie R. van Raaij, L. Basten Snoek, Jaap Bakker and Arjen Schots

To boost heterologous protein production the codon use of a gene of interest is often adapted to reflect the expression host’s codon use in highly expressed genes (optimal codons). However, the results obtained with this strategy are variable. A comparison between the overall codon use and the codon use in highly expressed genes of several plant species revealed that optimal codons are not always the codons of which the use is most increased with expression. Although the codon composition of highly expressed genes differs between monocots and dicots, often the same codons are linked strongest to expression (here after named expression codons). We used these conserved expression codons to optimise the codon composition of three genes, which enhanced protein yield significantly upon stable and transient expression in plants. Upon stable transformation both transcript levels and protein yield per transcript increased. Next, we analysed whether this expression-linked codon bias found in plants also extends to other kingdoms of life. Thereto, expression-linked codon use was investigated in Escherichia coli (Bacteria), Saccharomyces cerevisiae (Fungi), Caenorhabditis elegans (Animalia) and Mus musculus (Animalia) using more than 250 microarrays per species. We found that a common codon use bias exits over all species. In addition, computational analyses of various mRNA characteristics revealed a similar selection pressure across kingdoms that increases both stability and translatability. Combining gene expression data with available protein abundance data showed that an increased number of stem-loop transitions together with a reduction of stem size increases translation efficiency. An algorithm was developed that combines the use of optimal and expression codons to create an ideal mRNA structure for any given gene. This algorithm was again tested in plants and lead to a significant increase in protein production. The spin off company TripleT Biosciences combines this algorithm with all available knowledge on codon use and offers a codon optimization tool.

The PERKs of being resistant – on the exploitation of a novel source of nematode resistance in vegetable crops
Lozano Torres, J.L. ; Warmerdam, S. ; Diaz Granados Muñoz, A. ; Varossieau, K. ; Schaik, C.C. van; Raaij, D.R. van; Goverse, A. ; Smant, G. - \ 2016
The impact of plant-parasitic nematodes on food security has increased significantly over the past few years for two reasons. First, most of the pesticides currently used to control nematodes face a global ban, because of serious environmental and health concerns. Second, rising soil temperatures by global warming and genetic adaptations in nematode populations undermine the use of many major nematode resistance genes in food crops. At present, nematode resistance breeding in tomato and other vegetable crops is based on highly specific resistance genes, most of which appear to be temperature sensitive (>28° C). These circumstances demand the plant breeding industry to refocus on novel types of nematode resistance. However, very little is known of alternative sources of nematode resistance in plants.Recently, I demonstrated that nematodes are far more sensitive to basal host immunity than expected. Unlike highly specific major resistances, basal immunity is regulated in plant cells by surface-localized receptors. These receptors survey the cell exterior for tissue damage that betrays the presence of invaders. Plant parasitic nematodes modify plant cell walls during host invasion and feeding, but how this affects basal immunity is unknown. My thesis work provides first evidence that specific plant cell wall-associated receptors (i.e. PERKs) most likely mediate basal immunity to both cyst and root-knot nematodes. Therefore, the aim of this project is to elucidate the molecular mechanisms underlying the activation of damage-induced basal immunity to plant-parasitic nematodes. I will first use high-resolution transcriptomics to study Arabidopsis knockout mutants lacking these specific plant cell wall-associated receptors. This will enable me to further identify and characterize downstream mechanisms underlying basal immunity activated by nematode-induced damage in plants. Natural genetic variation in key regulators of damage-induced host defences can ultimately be exploited in breeding programs to develop novel cultivars with enhanced broad-spectrum basal resistance to nematodes.
The functional characterization of positively-selected effectors of the root-knot nematode Meloidogyne incognita
Verhoeven, Ava ; Prins, J.C.P. ; Finkers-Tomczak, A.M. ; Varossieau, K. ; Raaij, D.R. van; Pomp, H. ; Goverse, A. ; Smant, G. - \ 2016
Transient expression of secretory IgA in planta is optimal using a multi-gene vector and may be further enhanced by improving joining chain incorporation
Westerhof, L.B. ; Wilbers, R.H.P. ; Raaij, D.R. van; Wijk, Marloes van; Goverse, A. ; Bakker, J. ; Schots, A. - \ 2016
Frontiers in Plant Science 6 (2016). - ISSN 1664-462X - 12 p.
Secretory IgA (sIgA) is a crucial antibody in host defence at mucosal surfaces. It is a promising antibody isotype in a variety of therapeutic settings such as passive vaccination and treatment of inflammatory disorders. However, heterologous production of this heteromultimeric protein complex is still suboptimal. The challenge is the coordinate expression of the four required polypeptides; the alpha heavy chain, the light chain, the joining chain and part of the polymeric-Ig-receptor called the secretory component, in a 4:4:1:1 ratio. We evaluated the transient expression of three sIgAκ variants, harbouring the heavy chain isotype α1, α2m1 or α2m2, of the clinical antibody Ustekinumab in planta. Ustekinumab is directed against the p40 subunit that is shared by the pro-inflammatory cytokines interleukin (IL)-12 and IL-23. A sIgA variant of this antibody may enable localized treatment of inflammatory bowel disease. Of the three different sIgA variants we obtained the highest yield with sIgA1κ reaching up to 373 μg sIgA/ mg total soluble protein. The use of a multi-cassette vector containing all four expression cassettes was most efficient. However, not the expression strategy, but the incorporation of the joining chain turned out to be the limiting step for sIgA production. Our data demonstrate that transient expression in planta is suitable for the economic production of heteromultimeric protein complexes such as sIgA.
WHO European Childhood Obesity Surveillance Initiative: impact of type of clothing worn during anthropometric measurements and timing of the survey on weight and body mass index outcome measures in 6–9-year-old children
Wijnhoven, T.M.A. ; Raaij, J.M.A. van; Spinelli, A. ; Yngve, Agneta ; Lissner, L. ; Spiroski, I. ; Farrugia Sant Angelo, V. ; Pérez-Farinós, Napoleon ; Martos, E. ; Heinen, M. ; Kunesova, M. ; Rito, A.I. ; Hovengen, R. ; Starc, G. ; Duleva, Vesselka ; Pudule, I. ; Petrauskiene, Ausra ; Braeckevelt, L. ; Hassapidou, M. ; Breda, João ; Veer, P. van 't - \ 2016
Epidemiology Research International 2016 (2016). - ISSN 2090-2972 - 16 p.
The World Health Organization European Childhood Obesity Surveillance Initiative (COSI) conducted examinations in 6–9-year-old children from 16 countries in the first two rounds of data collection. Allowing participating countries to adhere to their local legal requirements or adapt to other circumstances required developing a flexible protocol for anthropometric
procedures.
Objectives
. (1) Review intercountry variation in types of clothing worn by children during weight and height measurements, clothes weight adjustments applied, timing of the survey, and duration of data collection; (2) assess the impact of the observed variation in these practices on the children’s weight or body mass index (BMI) outcome measures.
Results
.The relative difference between countries’ unadjusted and clothes-adjusted prevalence estimates for overweight was 0.3–11.5%; this figure was 1.4–33.3% for BMI-for-age Z -score values. Monthly fluctuations in mean BMI-for-age
Z -score values did not show a systematic seasonal effect. The majority of the monthly BMI-for-age Z-score values did not differ statistically within a country;
only 1–3 monthly values were statistically different within some countries.
Conclusions
. The findings of the present study suggest that the built-in flexibility in the COSI protocol concerning the data collection practices addressed in the paper can be kept and thus do not necessitate a revision of the COSI protocol.
Co-expression of the protease furin in Nicotiana benthamiana leads to efficient processing of latent transforming growth factor-b1 into a biologically active protein
Wilbers, R.H.P. ; Westerhof, L.B. ; Raaij, D.R. van; Adrichem, Marloes van; Prakasa, A.D. ; Lozano Torres, J.L. ; Bakker, J. ; Smant, G. ; Schots, A. - \ 2016
Plant Biotechnology Journal 14 (2016)8. - ISSN 1467-7644 - p. 1695 - 1704.
Transforming growth factor beta (TGF-β) is a signalling molecule that plays a key role in developmental and immunological processes in mammals. Three TGF-β isoforms exist in humans, and each isoform has unique therapeutic potential. Plants offer a platform for the production of recombinant proteins, which is cheap and easy to scale up and has a low risk of contamination with human pathogens. TGF-β3 has been produced in plants before using a chloroplast expression system. However, this strategy requires chemical refolding to obtain a biologically active protein. In this study, we investigated the possibility to transiently express active human TGF-β1 in Nicotiana benthamiana plants. We successfully expressed mature TGF-β1 in the absence of the latency-associated peptide (LAP) using different strategies, but the obtained proteins were inactive. Upon expression of LAP-TGF-β1, we were able to show that processing of the latent complex by a furin-like protease does not occur in planta. The use of a chitinase signal peptide enhanced the expression and secretion of LAP-TGF-β1, and co-expression of human furin enabled the proteolytic processing of latent TGF-β1. Engineering the plant post-translational machinery by co-expressing human furin also enhanced the accumulation of biologically active TGF-β1. This engineering step is quite remarkable, as furin requires multiple processing steps and correct localization within the secretory pathway to become active. Our data demonstrate that plants can be a suitable platform for the production of complex proteins that rely on specific proteolytic processing.
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