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.

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Apoplastic venom allergen-like proteins of plant-parasitic nematodes modulate the activation of plant innate immunity by cell surface receptors
Lozano Torres, J.L. ; Wilbers, R.H.P. ; Warmerdam, S. ; Varossieau, K. ; Willig, J. ; Schaik, C.C. van; Asojo, Oluwatoyin A. ; Darwiche, Rabih ; Schneiter, Roger ; Drurey, Claire ; Maizels, Rick M. ; Goverse, A. ; Schots, A. ; Smant, G. - \ 2018
Despite causing considerable damage to host tissue during parasitism, nematodes establish persistent infections in both animals and plants. An elaborate repertoire of nematode effectors modulates damage-triggered immune responses of the host. However, the nature and mode of action of most of nematode immunomodulatory compounds is not well understood. We discovered that the nematode effectors named the venom allergen-like proteins (VAPs) selectively suppress host immunity during the onset of parasitism in plants. VAPs are uniquely conserved in secretions of all animal- and plant-parasitic nematodes, but their role in parasitism has remained elusive. Knocking-down the expression of Gr-VAP1 hampered the infectivity of the potato cyst nematode Globodera rostochiensis. By contrast, heterologous expression of Gr-VAP1 and VAPs from the beet cyst nematode Heterodera schachtii, in Arabidopsis, resulted in the loss of basal immunity to multiple pathogens. Surprisingly, VAPs only affect the defence responses mediated by surface-localised immune receptors. The modulation of basal immunity by ectopic expression of VAPs involves extracellular protease-based host defences and jasmonic acid responses. Crystal structures of VAPs revealed lipid binding motifs. In these cavities VAPs can bind palmitate and sterol both in vitro and in vivo. The delivery of VAPs into host tissue coincides with large modifications in the extracellular matrix by migratory nematodes. We, therefore, conclude that parasitic nematodes most likely utilise VAPs to suppress the activation of defences by immunogenic breakdown products in damaged host tissue.
Frontiers in Plant Science (Journal)
Schots, Arjen - \ 2018
Frontiers in Plant Science (2018). - ISSN 1664-462X
Distinct Roles of Non-Overlapping Surface Regions of the Coiled-Coil Domain in the Potato Immune Receptor Rx1
Slootweg, Erik J. ; Spiridon, Laurentiu N. ; Martin, Eliza C. ; Tameling, Wladimir I.L. ; Townsend, Philip D. ; Pomp, Rikus ; Roosien, Jan ; Drawska, Olga ; Sukarta, Octavina C.A. ; Schots, Arjen ; Borst, Jan Willem ; Joosten, Matthieu H.A.J. ; Bakker, Jaap ; Smant, Geert ; Cann, Martin J. ; Petrescu, Andrei-Jose ; Goverse, Aska - \ 2018
Plant Physiology 178 (2018)3. - ISSN 0032-0889 - p. 1310 - 1331.
The intracellular immune receptor Rx1 of potato (Solanum tuberosum), which confers effector-triggered immunity to Potato virus X, consists of a central nucleotide-binding domain (NB-ARC) flanked by a carboxyl-terminal leucine-rich repeat (LRR) domain and an amino-terminal coiled-coil (CC) domain. Rx1 activity is strictly regulated by interdomain interactions between the NB-ARC and LRR, but the contribution of the CC domain in regulating Rx1 activity or immune signaling is not fully understood. Therefore, we used a structure-informed approach to investigate the role of the CC domain in Rx1 functionality.
Targeted mutagenesis of CC surface residues revealed separate regions required for the intramolecular and intermolecular interaction of the CC with the NB-ARC-LRR and the cofactor Ran GTPase-activating protein2 (RanGAP2), respectively. None of the mutant Rx1 proteins was constitutively active, indicating that the CC does not contribute to the autoinhibition of Rx1 activity. Instead, the CC domain acted as a modulator of downstream responses involved in effector-triggered immunity. Systematic disruption of the hydrophobic interface between the four helices of the CC enabled the uncoupling of cell death and disease resistance responses. Moreover, a strong dominant negative effect on Rx1-mediated resistance and cell death was observed upon coexpression of the CC alone with full-length Rx1 protein, which depended on the RanGAP2-binding surface of the CC. Surprisingly, coexpression of the N-terminal half of the CC enhanced Rx1-mediated resistance, which further indicated that the CC functions as a scaffold for downstream components involved in the modulation of disease resistance or cell death signaling.
Nicotiana benthamianaα-galactosidase A1.1 can functionally complement human α-galactosidase A deficiency associated with Fabry disease
Kytidou, Kassiani ; Beekwilder, Jules ; Artola, Marta ; Meel, Eline van; Wilbers, Ruud H.P. ; Moolenaar, Geri F. ; Goosen, Nora ; Ferraz, Maria J. ; Katzy, Rebecca ; Voskamp, Patrick ; Florea, Bogdan I. ; Hokke, Cornelis H. ; Overkleeft, Herman S. ; Schots, Arjen ; Bosch, Dirk ; Pannu, Navraj ; Aerts, Johannes M.F.G. - \ 2018
Journal of Biological Chemistry 293 (2018)26. - ISSN 0021-9258 - p. 10042 - 10058.

α-Galactosidases (EC 3.2.1.22) are retaining glycosidases that cleave terminal α-linked galactose residues from glycoconjugate substrates. α-Galactosidases take part in the turnover of cell wall-associated galactomannans in plants and in the lysosomal degradation of glycosphingolipids in animals. Deficiency of human α-galactosidase A (α-Gal A) causes Fabry disease (FD), a heritable, X-linked lysosomal storage disorder, characterized by accumulation of globotriaosylceramide (Gb3) and globotriaosylsphingosine (lyso-Gb3). Current management of FD involves enzyme-replacement therapy (ERT). An activitybased probe (ABP) covalently labeling the catalytic nucleophile of α-Gal A has been previously designed to study α-galactosidases for use in FD therapy. Here, we report that this ABP labels proteins in Nicotiana benthamiana leaf extracts, enabling the identification and biochemical characterization of an N. benthamiana α-galactosidase we name here A1.1 (gene accession ID GJZM-1660). The transiently overexpressed and purified enzyme was a monomer lacking N-glycans and was active toward 4-methylumbelliferyl-α-D-galactopyranoside substrate (Km = 0.17 mM) over a broad pH range. A1.1 structural analysis by X-ray crystallography revealed marked similarities with human α-Gal A, even including A1.1's ability to hydrolyze Gb3 and lyso-Gb3, which are not endogenous in plants. Of note, A1.1 uptake into FD fibroblasts reduced the elevated lyso-Gb3 levels in these cells, consistent with A1.1 delivery to lysosomes as revealed by confocal microscopy. The ease of production and the features of A1.1, such as stability over a broad pH range, combined with its capacity to degrade glycosphingolipid substrates, warrant further examination of its value as a potential therapeutic agent for ERT-based FD management.

Characterization of Schistosoma mansoni fucosyltransferases for glyco-engineering of 'native' helminth N-glycan structures in planta
Noort, Kim van; Nguyen, Dieu-Linh ; Hokke, C. ; Schots, A. ; Wilbers, R.H.P. - \ 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.
Frontiers in Plant Science (Journal)
Schots, Arjen - \ 2017
Frontiers in Plant Science (2017). - ISSN 1664-462X
GM-CSF negatively regulates early IL-10 mediated responses
Wilbers, R.H.P. ; Westerhof, L.B. ; Raaij, D.R. van; Smant, G. ; Bakker, J. ; Schots, A. - \ 2017
bioRxiv (2017). - 14 p.
Inflammatory disorders are becoming more prevalent in the Western world. Treatment of these diseases relies on the intervention in inflammatory responses thereby restoring immune homeostasis. One cytokine that has the potential to restore immune homeostasis is the anti-inflammatory cytokine interleukin-10 (IL-10). But until now IL-10 treatment has not been as successful as anticipated. A reason for this may be that IL-10 responsiveness depends on the environment of the inflamed tissue. In this study we describe that granulocyte-macrophage colony-stimulating factor (GM-CSF) is a key cytokine that negatively regulates IL-10-mediated responses. Dendritic cells differentiated from bone marrow with GM- CSF have a reduced ability to respond to IL-10. Dendritic cells are impaired in their up-regulation of IL-10-induced SOCS3 expression and are unable to suppress LPS-induced TNF-α expression at an early time point. Furthermore, GM-CSF treatment partially replicates this phenotype in macrophages. Surprisingly, GM-CSF seems to regulate IL-10 activity in macrophages without affecting STAT3 activation. Still, GM-CSF induces constitutive phosphorylation of glycogen synthase kinase 3β, a signalling component downstream of the PI3K/Akt pathway. Knowledge on the exact mechanism by which GM-CSF negatively regulates IL-10 activity could give novel insights on the integration of signal transduction pathways elicited by different cytokines. Ultimately this knowledge could provide us with new therapeutic strategies to treat inflammatory disorders.
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 ; Lenz, Laurel L. - \ 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.
Sequence exchange between R genes converts virus resistance into nematode resistance, and vice versa
Slootweg, Erik J. ; Koropacka, K.B. ; Roosien, Jan ; Dees, Robert ; Overmars, Hein ; Klein Lankhorst, Rene ; Schaik, Casper van; Pomp, Rikus ; Bouwman-Smits, Liesbeth ; Helder, Hans ; Schots, Arjen ; Bakker, Jaap ; Smant, Geert ; Goverse, Aska - \ 2017
Plant Physiology 175 (2017). - ISSN 0032-0889 - p. 498 - 510.
Plants have evolved a limited repertoire of NB-LRR disease resistance (R) genes to protect themselves against a myriad of pathogens. This limitation is thought to be counterbalanced by the rapid evolution of NB-LRR proteins, as only few sequence changes have been shown to be sufficient to alter resistance specificities towards novel strains of a pathogen. However, little is known about the flexibility of NB-LRR genes to switch resistance specificities between phylogenetically unrelated pathogens. To investigate this, we created domain swaps between the close homologs Gpa2 and Rx1, which confer resistance in potato to the cyst nematode Globodera pallida and Potato virus X (PVX), respectively. The genetic fusion of the CC-NB-ARC of Gpa2 with the LRR of Rx1 (Gpa2CN/Rx1L) resulted in autoactivity, but lowering the protein levels restored its specific activation response including extreme resistance to PVX in potato shoots. The reciprocal construct (Rx1CN/Gpa2L) showed a loss-of-function phenotype, but exchange of the first 3 LRR repeats of Rx1 was sufficient to regain a wild type resistance response to G. pallida in the roots. These data demonstrate that exchanging the recognition moiety in the LRR is sufficient to convert extreme virus resistance in the leaves into mild nematode resistance in the roots, and vice versa. In addition, we show that the CC-NB-ARC can operate independently of the recognition specificities defined by the LRR domain, either above or belowground. These data show the versatility of NB-LRR genes to generate resistances to unrelated pathogens with completely different lifestyles and routes of invasion.
Schistosome egg antigens, including the glycoprotein IPSE/alpha-1, trigger the development of regulatory B cells
Haeberlein, Simone ; Obieglo, Katja ; Ozir-Fazalalikhan, Arifa ; Chayé, Mathilde A.M. ; Veninga, Henrike ; Vlugt, Luciën E.P.M. van der; Voskamp, Astrid ; Boon, Louis ; Haan, Joke M.M. den; Westerhof, Lotte B. ; Wilbers, Ruud H.P. ; Schots, Arjen ; Schramm, Gabriele ; Hokke, Cornelis H. ; Smits, Hermelijn H. - \ 2017
PLoS Pathogens 13 (2017)7. - ISSN 1553-7366
Infection with the helminth Schistosoma (S.) mansoni drives the development of interleukin (IL)-10-producing regulatory B (Breg) cells in mice and man, which have the capacity to reduce experimental allergic airway inflammation and are thus of high therapeutic interest. However, both the involved antigen and cellular mechanisms that drive Breg cell development remain to be elucidated. Therefore, we investigated whether S. mansoni soluble egg antigens (SEA) directly interact with B cells to enhance their regulatory potential, or act indirectly on B cells via SEA-modulated macrophage subsets. Intraperitoneal injections of S. mansoni eggs or SEA significantly upregulated IL-10 and CD86 expression by marginal zone B cells. Both B cells as well as macrophages of the splenic marginal zone efficiently bound SEA in vivo, but macrophages were dispensable for Breg cell induction as shown by macrophage depletion with clodronate liposomes. SEA was internalized into acidic cell compartments of B cells and induced a 3-fold increase of IL-10, which was dependent on endosomal acidification and further enhanced by CD40 ligation. IPSE/alpha-1, one of the major antigens in SEA, was also capable of inducing IL-10 in naïve B cells, which was reproduced by tobacco plant-derived recombinant IPSE. Other major schistosomal antigens, omega-1 and kappa-5, had no effect. SEA depleted of IPSE/alpha-1 was still able to induce Breg cells indicating that SEA contains more Breg cell-inducing components. Importantly, SEA- and IPSE-induced Breg cells triggered regulatory T cell development in vitro. SEA and recombinant IPSE/alpha-1 also induced IL-10 production in human CD1d+B cells. In conclusion, the mechanism of S. mansoni-induced Breg cell development involves a direct targeting of B cells by SEA components such as the secretory glycoprotein IPSE/alpha-1.
productie van medicinale eiwitten in tabak
Schots, Arjen - \ 2017
De comeback van tabak als medicijn
Schots, Arjen - \ 2017
Human Alpha Galactosidases Transiently Produced in Nicotiana benthamiana Leaves: New Insights in Substrate Specificities with Relevance for Fabry Disease
Kytidou, Kassiani ; Beenakker, Thomas J.M. ; Westerhof, Lotte B. ; Hokke, Cornelis H. ; Moolenaar, Geri F. ; Goosen, Nora ; Mirzaian, Mina ; Ferraz, Maria J. ; Geus, Mark De; Kallemeijn, Wouter W. ; Overkleeft, Herman S. ; Boot, Rolf G. ; Schots, Arjen ; Bosch, Dirk ; Aerts, Johannes M.F.G. - \ 2017
Frontiers in Plant Science 8 (2017). - ISSN 1664-462X
Deficiency of a-galactosidase A (a-GAL) causes Fabry disease (FD), an X-linked storage disease of the glycosphingolipid globtriaosylcerammide (Gb3) in lysosomes of various cells and elevated plasma globotriaosylsphingosine (Lyso-Gb3) toxic for podocytes and nociceptive neurons. Enzyme replacement therapy is used to treat the disease, but clinical efficacy is limited in many male FD patients due to development of neutralizing antibodies (Ab). Therapeutic use of modified lysosomal a-N-acetyl-galactosaminidase (a-NAGAL) with increased a-galactosidase activity (a-NAGALEL) has therefore been suggested. We transiently produced in Nicotiana benthamiana leaves functional a-GAL,
a-NAGAL, and a-NAGALEL enzymes for research purposes. All enzymes could be
visualized with activity-based probes covalently binding in their catalytic pocket.
Characterization of purified proteins indicated that a-NAGALEL is improved in activity toward artificial 4MU-a-galactopyranoside. Recombinant a-NAGALEL and a-NAGAL are not neutralized by Ab-positive FD serum tested and are more stable in human plasma than a-GAL. Both enzymes hydrolyze the lipid substrates Gb3 and Lyso-Gb3 accumulating in Fabry patients. The addition to FD sera of a-NAGALEL, and to a lesser extent that of a-NAGAL, results in a reduction of the toxic Lyso-Gb3. In conclusion, our study suggests that modified a-NAGALEL might reduce excessive Lyso-Gb3 in FD serum. This neo-enzyme can be produced in Nicotiana benthamiana and might be further developed for the treatment of FD aiming at reduction of circulating Lyso-Gb3.
De comeback van tabak als medicijn
Schots, Arjen - \ 2017
Production and glyco-engineering of immunomodulatory helminth glycoproteins in plants
Wilbers, Ruud H.P. ; Westerhof, Lotte B. ; Noort, Kim Van; Obieglo, Katja ; Driessen, Nicole N. ; Everts, Bart ; Gringhuis, Sonja I. ; Schramm, Gabriele ; Goverse, Aska ; Smant, Geert ; Bakker, Jaap ; Smits, Hermelijn H. ; Yazdanbakhsh, Maria ; Schots, Arjen ; Hokke, Cornelis H. - \ 2017
Scientific Reports 7 (2017). - ISSN 2045-2322
Helminth parasites control host-immune responses by secreting immunomodulatory glycoproteins. Clinical trials and mouse model studies have demonstrated the potential of helminth-derived glycoproteins for the treatment of immune-related diseases, like allergies and autoimmune diseases. Studies are however hampered by the limited availability of native parasite-derived proteins. Moreover, recombinant protein production systems have thus far been unable to reconstitute helminth-like glycosylation essential for the functionality of some helminth glycoproteins. Here we exploited the flexibility of the N-glycosylation machinery of plants to reconstruct the helminth glycoproteins omega-1 and kappa-5, two major constituents of immunomodulatory Schistosoma mansoni soluble egg antigens. Fine-tuning transient co-expression of specific glycosyltransferases in Nicotiana benthamiana enabled the synthesis of Lewis X (LeX) and LDN/LDN-F glycan motifs as found on natural omega-1 and kappa-5, respectively. In vitro and in vivo evaluation of the introduction of native LeX motifs on plant-produced omega-1 confirmed that LeX on omega-1 contributes to the glycoprotein’s Th2-inducing properties. These data indicate that mimicking the complex carbohydrate structures of helminths in plants is a promising strategy to allow targeted evaluation of therapeutic glycoproteins for the treatment of inflammatory disorders. In addition, our results offer perspectives for the development of effective anti-helminthic vaccines by reconstructing native parasite glycoprotein antigens.
Mimicking immunomodulatory helminth glycoproteins in plants to enable treatment of inflammatory diseases
Wilbers, R.H.P. ; Westerhof, L.B. ; Noort, Kim van; Obieglo, K. ; Driessen, N.N. ; Everts, B. ; Goverse, A. ; Smant, G. ; Bakker, J. ; Smits, H.H. ; Yazdanbakhsh, M. ; Schots, A. ; Hokke, C.H. - \ 2017
Helminth parasites control host-immune responses by secreting immunomodulatory glycoproteins. Clinical trials and mouse model studies have demonstrated the potential of helminths and helminth-derived glycoproteins for the treatment of immune-related diseases, like allergies and autoimmune diseases. Studies are however hampered by the limited availability of native parasite-derived proteins. Moreover, recombinant protein production systems have thus far been unable to reconstitute helminth-like glycosylation essential for the functionality of helminth glycoproteins. Here we exploited the flexibility of the N-glycosylation machinery of plants to reconstruct two helminth glycoproteins, omega-1 and kappa-5, major constituents of immunomodulatory Schistosoma mansoni soluble egg antigens. Fine-tuning transient co-expression of specific glycosyltransferases in Nicotiana benthamiana enabled the synthesis of Lewis X (LeX) and LDN/LDN-F glycan motifs as found on natural omega-1 and kappa-5, respectively. In vitro and in vivo evaluation of the introduction of native LeX motifs on plant-produced omega-1 confirmed that LeX on omega-1 contributes to the glycoprotein’s Th2-inducing properties. These data indicate that mimicking the complex carbohydrate structures of helminths in plants is a promising strategy to allow targeted evaluation of therapeutic glycoproteins for the treatment of inflammatory disorders. In addition, our results offer perspectives for the development of effective anti-helminthic vaccines by reconstructing native parasite glycoprotein antigens.
Apoplastic Venom Allergen-like Proteins of Plant Parasitic Nematodes Modulate the Activation of Plant Innate Immunity by Cell Surface Receptors
Lozano Torres, J.L. ; Wilbers, R.H.P. ; Warmerdam, S. ; Finkers-Tomczak, A.M. ; Diaz Granados Muñoz, A. ; Schaik, C.C. van; Helder, J. ; Bakker, J. ; Goverse, A. ; Schots, A. ; Smant, G. - \ 2016
Despite causing considerable damage to host tissue during parasitism, nematodes establish persistent infections in both animals and plants. It is thought that an elaborate repertoire of nematode effectors suppress damage-triggered immune responses of the host. However, the nature and mode of action of most of nematode immunomodulatory compounds are not well understood. We have recently discovered that the effector venom allergen-like proteins (VAPs) of plant-parasitic nematodes selectively suppress host immunity during the onset of parasitism. VAPs are uniquely conserved in secretions of all animal- and plant-parasitic nematodes, but their role in parasitism has remained elusive. Knocking-down the expression of Gr-VAP1 severely hampered the infectivity of Globodera rostochiensis. By contrast, heterologous expression of Gr-VAP1 and VAPs from Heterodera schachtii in plants resulted in the loss of basal immunity to multiple pathogens. Surprisingly, these VAPs only affect the defense responses mediated by surface-localized immune receptors. The modulation of basal immunity by ectopic VAPs involves extracellular protease-based host defenses and chloroplast-localized non-photochemical quenching. The delivery of VAPs into host tissue coincides with the enzymatic breakdown of plant cell walls by migratory nematodes. We, therefore, conclude that parasitic nematodes most likely utilize VAPs to suppress the activation of defenses by immunogenic breakdown products in damaged host tissue.
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.
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.
Characterization of Schistosoma mansoni fucosyltransferases for glyco-engineering of ‘native’ helminth N-glycan structures in planta
Noort, Kim van; Wilbers, R.H.P. ; Westerhof, L.B. ; Hokke, C.H. ; Smant, G. ; Bakker, J. ; Schots, A. - \ 2016
Secretory glycoproteins of parasitic helminths are in the spotlight as biopharmaceuticals because of their strong, glycan-dependent immunomodulatory properties. Helminths and their secretions have been shown to dampen allergic reactions and autoimmune disorders, such as inflammatory bowel diseases, multiple sclerosis and rheumatoid arthritis. Clinical trials with live parasites and mouse model studies with excretory/secretory proteins are promising and reveal an urgent need for the large-scale production of defined secretory glycoproteins from helminths. Helminth N-glycans contribute to immunomodulation, but have unique structures that cannot be synthesized in current biopharmaceutical production systems. The trematode Schistosoma mansoni produces complex highly fucosylated N-glycan structures on its glycoproteins. Therefore, modifications of the N-glycosylation machinery of the expression host are required for the production of S. mansoni--derived immunomodulatory glycoproteins with their native N-glycans. This can be achieved by knocking-in or knocking-out specific glycosyltransferases, allowing synthesis of specific helminth N-glycan structures. Plants are remarkable versatile as glyco-engineering platform for the synthesis of glycoproteins with tailored N-glycans. However, for the synthesis of highly fucosylated N-glycans from S. mansoni knowledge is lacking on how these specific structures are synthesized. For this purpose, we examined the function of ten selected fucosyltransferases from Schistosoma mansoni using transient co-expression with immunomodulatory omega-1 as a model protein. Two fucosyltransferases were identified that specifically couple fucoses to the core glucosamine with an α1,3 or α1,6 bond. These two fucosyltransferases can be used to obtain α1,3- and α1,6 core fucosylated N-glycan structures found on native helminth secreted immunomodulatory proteins. Our results show the versatility of plants both as a production system and as a system for characterizing glycosyltransferase functionality. Further characterization of S. mansoni fucosyltransferases and other glycosyltransferases will expand our glyco-engineering toolbox and offers perspectives for the synthesis of novel complex helminth N-glycan structures in plants.
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