Functional characterization of Schistosoma mansoni fucosyltransferases in Nicotiana benthamiana plants

Helminth parasites secrete a wide variety of immunomodulatory proteins and lipids to dampen host immune responses. Many of these immunomodulatory compounds are modified with complex sugar structures (or glycans), which play an important role at the host–parasite interface. As an example, the human blood fluke Schistosoma mansoni produces highly fucosylated glycan structures on glycoproteins and glycolipids. Up to 20 different S. mansoni fucosyltransferase (SmFucT) genes can be found in genome databases, but thus far only one enzyme has been functionally characterized. To unravel the synthesis of highly fucosylated N-glycans by S. mansoni, we examined the ability of ten selected SmFucTs to modify N-glycans upon transient expression in Nicotiana benthamiana plants. All enzymes were localized in the plant Golgi apparatus, which allowed us to identify the SmFucTs involved in core fucosylation and the synthesis of complex antennary glycan motifs. This knowledge provides a starting point for investigations into the role of specific fucosylated glycan motifs of schistosomes in parasite-host interactions. The functionally characterized SmFucTs can also be applied to synthesize complex N-glycan structures on recombinant proteins to study their contribution to immunomodulation. Furthermore, this plant expression system will fuel the development of helminth glycoproteins for pharmaceutical applications or novel anti-helminth vaccines

Glyco-Engineering Plants to Produce Helminth Glycoproteins as Prospective Biopharmaceuticals: Recent Advances, Challenges and Future Prospects

Glycoproteins are the dominant category among approved biopharmaceuticals, indicating their importance as therapeutic proteins. Glycoproteins are decorated with carbohydrate structures (or glycans) in a process called glycosylation. Glycosylation is a post-translational modification that is present in all kingdoms of life, albeit with differences in core modifications, terminal glycan structures, and incorporation of different sugar residues. Glycans play pivotal roles in many biological processes and can impact the efficacy of therapeutic glycoproteins. The majority of biopharmaceuticals are based on human glycoproteins, but non-human glycoproteins, originating from for instance parasitic worms (helminths), form an untapped pool of potential therapeutics for immune-related diseases and vaccine candidates. The production of sufficient quantities of correctly glycosylated putative therapeutic helminth proteins is often challenging and requires extensive engineering of the glycosylation pathway. Therefore, a flexible glycoprotein production system is required that allows straightforward introduction of heterologous glycosylation machinery composed of glycosyltransferases and glycosidases to obtain desired glycan structures. The glycome of plants creates an ideal starting point for N- and O-glyco-engineering of helminth glycans. Plants are also tolerant toward the introduction of heterologous glycosylation enzymes as well as the obtained glycans. Thus, a potent production platform emerges that enables the production of recombinant helminth proteins with unusual glycans. In this review, we discuss recent advances in plant glyco-engineering of potentially therapeutic helminth glycoproteins, challenges and their future prospects

Re-evaluation of IL-10 signaling reveals novel insights on the contribution of the intracellular domain of the IL-10R2 chain

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

Re-evaluation of IL-10 signaling reveals novel insights on the contribution of the intracellular domain of the IL-10R2 chain

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

Granulocyte-macrophage colony-stimulating factor negatively regulates early IL-10-mediated responses

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

Crystal structure of Brugia malayi venom allergen-like protein-1 (BmVAL-1), a vaccine candidate for lymphatic filariasis

Brugia malayi is a causative agent of lymphatic filariasis, a major tropical disease. The infective L3 parasite stage releases immunomodulatory proteins including the venom allergen-like proteins (VALs), which are members of the SCP/TAPS (Sperm-coating protein/Tpx/antigen 5/pathogenesis related-1/Sc7) superfamily. BmVAL-1 is a major target of host immunity with >90% of infected B. malayi microfilaraemic cases being seropositive for antibodies to BmVAL-1. This study is part of ongoing efforts to characterize the structures and functions of important B. malayi proteins. Recombinant BmVAL-1 was produced using a plant expression system, crystallized and the structure was solved by molecular replacement and refined to 2.1 Å, revealing the characteristic alpha/beta/alpha sandwich topology of eukaryotic SCP/TAPS proteins. The protein has more than 45% loop regions and these flexible loops connect the helices and strands, which are longer than predicted based on other parasite SCP/TAPS protein structures. The large central cavity of BmVAL-1 is a prototypical CRISP cavity with two histidines required to bind divalent cations. The caveolin-binding motif (CBM) that mediates sterol binding in SCP/TAPS proteins is large and open in BmVAL-1 and is N-glycosylated. N-glycosylation of the CBM does not affect the ability of BmVAL-1 to bind sterol in vitro. BmVAL-1 complements the in vivo sterol export phenotype of yeast mutants lacking their endogenous SCP/TAPS proteins. The in vitro sterol-binding affinity of BmVAL-1 is comparable with Pry1, a yeast sterol transporting SCP/TAPS protein. Sterol binding of BmVAL-1 is dependent on divalent cations. BmVAL-1 also has a large open palmitate-binding cavity, which binds palmitate comparably to tablysin-15, a lipid-binding SCP/TAPS protein. The central cavity, CBM and palmitatebinding cavity of BmVAL-1 are interconnected within the monomer with channels that can serve as pathways for water molecules, cations and small molecules

β-Hexosaminidases Along the Secretory Pathway of Nicotiana benthamiana Have Distinct Specificities Toward Engineered Helminth N-Glycans on Recombinant Glycoproteins

Secretions of parasitic worms (helminths) contain a wide collection of immunomodulatory glycoproteins with the potential to treat inflammatory disorders, like autoimmune diseases. Yet, the identification of single molecules that can be developed into novel biopharmaceuticals is hampered by the limited availability of native parasite-derived proteins. Recently, pioneering work has shown that helminth glycoproteins can be produced transiently in Nicotiana benthamiana plants while simultaneously mimicking their native helminth N-glycan composition by co-expression of desired glycosyltransferases. However, efficient “helminthization” of N-glycans in plants by glyco-engineering seems to be hampered by the undesired truncation of complex N-glycans by β-N-acetyl-hexosaminidases, in particular when aiming for the synthesis of N-glycans with antennary GalNAcβ1-4GlcNAc (LacdiNAc or LDN). In this study, we cloned novel β-hexosaminidase open reading frames from N. benthamiana and characterized the biochemical activity of these enzymes. We identified HEXO2 and HEXO3 as enzymes responsible for the cleavage of antennary GalNAc residues of N-glycans on the model helminth glycoprotein kappa-5. Furthermore, we reveal that each member of the HEXO family has a distinct specificity for N-glycan substrates, where HEXO2 has strict β-galactosaminidase activity, whereas HEXO3 cleaves both GlcNAc and GalNAc. The identification of HEXO2 and HEXO3 as major targets for LDN cleavage will enable a targeted genome editing approach to reduce undesired processing of these N-glycans. Effective knockout of these enzymes could allow the production of therapeutically relevant glycoproteins with tailor-made helminth N-glycans in plants

Structural insights into a cooperative switch between one and two FimH bacterial adhesins binding pauci- and high-mannose type N-glycan receptors

The FimH type-1 fimbrial adhesin allows pathogenic Escherichia coli to adhere to glycoproteins in the epithelial linings of human bladder and intestinal tract, by using multiple fimbriae simultaneously. Pauci- and high-mannose type N-glycans are natural FimH receptors on those glycoproteins. Oligomannose-3 and oligomannose-5 bind with the highest affinity to FimH by using the same Manα1,3Man branch. Oligomannose-6 is generated from oligomannose-5 in the next step of the biogenesis of high-mannose N-glycans, by the transfer of a mannose in α1,2-linkage onto this branch. Using serial crystallography and by measuring the kinetics of binding, we demonstrate that shielding the high-affinity epitope drives the binding of multiple FimH molecules. First, we profiled FimH glycan binding on a microarray containing paucimannosidic N-glycans and in a FimH LEctPROFILE assay. To make the transition to oligomannose-6, we measured the kinetics of FimH binding using paucimannosidic N-glycans, glycoproteins and all four α-dimannosides conjugated to bovine serum albumin. Equimolar mixed interfaces of the dimannosides present in oligomannose-6 and molecular dynamics simulations suggest a positive cooperativity in the bivalent binding of Manα1,3Manα1 and Manα1,6Manα1 dimannosides. The binding of core α1,6-fucosylated oligomannose-3 in cocrystals of FimH is monovalent but interestingly the GlcNAc1—Fuc moiety retains highly flexibility. In cocrystals with oligomannose-6, two FimH bacterial adhesins bind the Manα1,3Manα1 and Manα1,6Manα1 endings of the second trimannose core (A-4′-B). This cooperative switch towards bivalent binding appears sustainable beyond a molar excess of oligomannose-6. Our findings provide important novel structural insights for the design of multivalent FimH antagonists that bind with positive cooperativity

The N-glycan on Asn54 affects the atypical N-glycan composition of plant-produced interleukin-22, but does not influence its activity

Host-parasite interactio