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, A.; Smant, G.; Bakker, J.; Smits, Hermelijn H.; Yazdanbakhsh, Maria; Schots, A.; Hokke, Cornelis H.

doi:10.1038/srep45910

Cited by 55 publications

(95 citation statements)

References 33 publications

(56 reference statements)

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“…Despite the enormous success, overexpression of proteins for glyco‐modulation is not always straightforward. There are examples in the literature reporting drawbacks of gene overexpression including alterations on plant development and mislocalization of the target protein …”

Section: Introductionmentioning

confidence: 99%

Promoter Choice Impacts the Efficiency of Plant Glyco‐Engineering

Kallolimath

Gruber

Steinkellner

et al. 2017

Biotechnology Journal

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Glyco-modulation of therapeutic proteins produced in plants has shown great success. Plant-based expression platforms for tailored human-like N-glycosylation are based on the overexpression of foreign genes. However, drawbacks such as protein miss targeting, interference with endogenous glycosyltransferases, or with plant development hamper the widespread use of the technology. Here a technique that facilitates the generation of recombinant proteins with targeted N-glycosylation at high homogeneity is described. It is focused on the synthesis of human-type b1,4-galactosylation by the overexpression of the human b1,4-galactosyltransferase (GalT) in Nicotiana benthamiana. A GalT construct that targets the enzyme to the required late Golgi compartment ( ST GalT) is transiently co-expressed with two pharmaceutically relevant glycoproteins. The impact of eight promoters driving the expression of ST GalT is evaluated by mass spectrometry (MS) -based analyses. It is shown that five promoters (amongst them high expressors) induce aberrant non-human glycosylation. In contrast, three promoters, considered as moderately active, regulate gene expression to levels leading to an improved efficiency of di-galactosylation (and subsequent sialylation) on the reporter proteins. The results point to the importance of promoter choice for optimizing glycan engineering processes.

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Section: Introductionmentioning

confidence: 99%

Promoter Choice Impacts the Efficiency of Plant Glyco‐Engineering

Kallolimath

Gruber

Steinkellner

et al. 2017

Biotechnology Journal

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“…Parasitic helminths secrete immunomodulatory with certain N-glycan epitopes including Lewis X and LDN-F glycan motives that find importance in treatment of allergies and autoimmune diseases. Overexpression of glycosyltransferases including FucTs, GalTs, and GalNAcTs in N. benthamiana enabled the reconstruction of Lewis X and LDN-F motives [73] that find importance in the development of anti-helminthic vaccines.…”

Section: Introducing Helminth Glycosylation Into Plantsmentioning

confidence: 99%

Sialoglycans and genetically engineered plants

Ghosh¹

2020

Sialic Acids and Sialoglycoconjugates in the Biology of Life, Health and Disease

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“…In viable eggs, about 30 of the SEA proteins are located outside the developing miracidium and encompass the complement of secreted antigens (egg-secreted proteins, ESP) that interact with host tissues to facilitate the passage of the egg from the mesenteric veins to the intestinal lumen (27). The T2 ribonuclease omega-1 (ω1) is the principal Th2-inducing component of ESP with its Th2-polarizing activity dependent upon both its RNase activity and glycosylation (19, 20, 28). This RNase is hepatotoxic (29), and its secretion by eggs into the granuloma regulates the pattern recognition receptor signaling pathways in dendritic cells that, in turn, prime Th2 responses from CD4 + T cells (30).…”

Section: Introductionmentioning

confidence: 99%

Programmed genome editing of the omega-1 ribonuclease 1 of the blood fluke,Schistosoma mansoni

Ittiprasert

Mann

Karinshak

et al. 2018

Preprint

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34 35 CRISPR/Cas9 based genome editing has not yet been reported in schistosomes. Here, we tested 36 this approach by targeting omega-1 (ω1) of Schistosoma mansoni as a proof of principle. This 37 secreted ribonuclease is crucial for Th2 priming and granuloma formation, providing informative 38 immuno-pathological readouts for programmed genome editing. Schistosome eggs were either 39 exposed to recombinant Cas9 complexed with a synthetic guide RNA (sgRNA) complementary 40 to exon 6 of ω1 by electroporation or transduced with pseudotyped lentivirus encoding Cas9 and 41 the sgRNA. Some eggs were also transduced with a single stranded oligodeoxynucleotide donor 42 transgene that encoded six stop codons, flanked by 50 nt-long 5'-and 3'-microhomology arms 43 matching the predicted Cas9-catalyzed double stranded break (DSB) within ω1. CRISPResso 44 analysis of amplicons spanning the DSB revealed ~4.5% of the reads were mutated by insertions, 45deletions and/or substitutions, with an efficiency for homology directed repair of 0.19% insertion 46 of the donor transgene. Transcripts encoding ω1 were reduced >80%, and lysates of ω1-edited 47 eggs displayed diminished ribonuclease activity indicative that programmed editing mutated the 48 ω1 gene. Whereas soluble lysates of wild type eggs polarized Th2 cytokine responses including 49 IL-4 and IL-5 in human macrophage/T cell co-cultures, diminished levels of these cytokines 50followed the exposure to those of ω1-mutated schistosome eggs. Following injection of 51 schistosome eggs into the tail vein of BALB/c mice, the volume of pulmonary granulomas 52surrounding ω1-mutated eggs was 18-fold smaller than wild type eggs. Programmed genome 53 editing was active in schistosomes, Cas9-catalyzed chromosomal breakage was repaired by 54 homology directed repair and/or non-homologous end joining, and mutation of ω1 impeded the 55 capacity of schistosome eggs both to drive Th2 polarization and to provoke formation of 56 pulmonary circumoval granulomas. Knock-out of ω1 and the impaired immunological phenotype 57showcase the novel application of programmed gene editing in and functional genomics for 58 schistosomes. 59 60 61 Introduction 62 63Schistosomiasis is considered the most problematic of the human helminth diseases in terms of 64 morbidity and mortality [1][2][3][4]. The past decade has seen major advances in knowledge and 65 understanding of the pathophysiology, developmental biology, evolutionary relationships and 66 genome annotation of the human schistosomes [5][6][7][8][9][10][11][12][13][14][15][16]. Establishing CRISPR/Cas9 genome 67 editing in schistosomiasis would greatly enable effective functional genomics approaches. The 68 stable CRISPR/Cas9-based site-specific gene mutation and phenotyping will drive innovation 69 and a deeper understanding of schistosome pathogenesis, biology and evolution [17]. 70 71The schistosome egg plays a central role both in disease transmission and pathogenesis [1]. The 72 appearance of S. mansoni eggs in host tissues by six to seven week...

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Production and glyco-engineering of immunomodulatory helminth glycoproteins in plants

Cited by 55 publications

References 33 publications

Promoter Choice Impacts the Efficiency of Plant Glyco‐Engineering

Promoter Choice Impacts the Efficiency of Plant Glyco‐Engineering

Sialoglycans and genetically engineered plants

Programmed genome editing of the omega-1 ribonuclease 1 of the blood fluke,Schistosoma mansoni

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