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• W2026760618 abstract "Many therapeutic proteins are glycosylated and require terminal sialylation to attain full biological activity. Current manufacturing methods based on mammalian cell culture allow only limited control of this important posttranslational modification, which may lead to the generation of products with low efficacy. Here we report in vivo protein sialylation in plants, which have been shown to be well suited for the efficient generation of complex mammalian glycoproteins. This was achieved by the introduction of an entire mammalian biosynthetic pathway in Nicotiana benthamiana, comprising the coordinated expression of the genes for (i) biosynthesis, (ii) activation, (iii) transport, and (iv) transfer of Neu5Ac to terminal galactose. We show the transient overexpression and functional integrity of six mammalian proteins that act at various stages of the biosynthetic pathway and demonstrate their correct subcellular localization. Co-expression of these genes with a therapeutic glycoprotein, a human monoclonal antibody, resulted in quantitative sialylation of the Fc domain. Sialylation was at great uniformity when glycosylation mutants that lack plant-specific N-glycan residues were used as expression hosts. Finally, we demonstrate efficient neutralization activity of the sialylated monoclonal antibody, indicating full functional integrity of the reporter protein. We report for the first time the incorporation of the entire biosynthetic pathway for protein sialylation in a multicellular organism naturally lacking sialylated glycoconjugates. Besides the biotechnological impact of the achievement, this work may serve as a general model for the manipulation of complex traits into plants. Many therapeutic proteins are glycosylated and require terminal sialylation to attain full biological activity. Current manufacturing methods based on mammalian cell culture allow only limited control of this important posttranslational modification, which may lead to the generation of products with low efficacy. Here we report in vivo protein sialylation in plants, which have been shown to be well suited for the efficient generation of complex mammalian glycoproteins. This was achieved by the introduction of an entire mammalian biosynthetic pathway in Nicotiana benthamiana, comprising the coordinated expression of the genes for (i) biosynthesis, (ii) activation, (iii) transport, and (iv) transfer of Neu5Ac to terminal galactose. We show the transient overexpression and functional integrity of six mammalian proteins that act at various stages of the biosynthetic pathway and demonstrate their correct subcellular localization. Co-expression of these genes with a therapeutic glycoprotein, a human monoclonal antibody, resulted in quantitative sialylation of the Fc domain. Sialylation was at great uniformity when glycosylation mutants that lack plant-specific N-glycan residues were used as expression hosts. Finally, we demonstrate efficient neutralization activity of the sialylated monoclonal antibody, indicating full functional integrity of the reporter protein. We report for the first time the incorporation of the entire biosynthetic pathway for protein sialylation in a multicellular organism naturally lacking sialylated glycoconjugates. Besides the biotechnological impact of the achievement, this work may serve as a general model for the manipulation of complex traits into plants. IntroductionThe outstanding specificity of therapeutic glycoproteins places them among the fastest growing class of pharmaceutical products. Many of these drugs need terminal sialylation, the final and most complex step of human N-glycosylation, for optimal therapeutic potency. Therefore manufacturing is currently restricted to mammalian cell-based systems that are able to perform this important posttranslational modification, although with major limitations. The naturally present glycosylation repertoire of mammalian host cells promotes the generation of a number of different terminal structures and thus leads to heterogeneous glycosylation. Moreover, these modifications may differ from the authentic human glycosylation. For example, CHO 4The abbreviations used are: CHOChinese hamster ovaryGalTβ1,4-galactosyltransferaseST-GalTmodified β1,4-galactosyltransferaseSTα2,6-sialyltransferaseCMASCMP-N-acetylneuraminic acid synthetaseNANSN-acetylneuraminic acid phosphate synthaseGNEUDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinaseCSTCMP-Neu5Ac transportermAbmonoclonal antibodyHIVhuman immunodeficiency virusGFPgreen fluorescent proteinRubiscoribulose-bisphosphate carboxylase/oxygenaseWTwild typeMES4-morpholineethanesulfonic acidMSmass spectrometryMS/MStandem MSLCliquid chromatographyESIelectrospray mass ionizationTOFtime-of-flight. cells, widely used for the expression of recombinant glycoproteins, do not naturally produce human type α2,6-sialylation. These shortcomings in many cases lead to drugs with reduced biological potency, of which recombinant hormones such as human erythropoietin and interferons are prominent examples (1Bork K. Horstkorte R. Weidemann W. J. Pharm. Sci. 2009; 98: 3499-3508Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). In addition, about 10% of the N-glycans in the Fc region of human serum IgG is sialylated (2Stadlmann J. Pabst M. Kolarich D. Kunert R. Altmann F. Proteomics. 2008; 8: 2858-2871Crossref PubMed Scopus (262) Google Scholar). Although the anti-inflammatory effects of sialylated IgG have recently been demonstrated (3Anthony R.M. Nimmerjahn F. Ashline D.J. Reinhold V.N. Paulson J.C. Ravetch J.V. Science. 2008; 320: 373-376Crossref PubMed Scopus (637) Google Scholar), very little information on the impact of this common modification at the molecular level is available (4Scallon B.J. Tam S.H. McCarthy S.G. Cai A.N. Raju T.S. Mol. Immunol. 2007; 44: 1524-1534Crossref PubMed Scopus (317) Google Scholar). This explains the general interest in novel expression hosts and strategies to engineer glycosylation not only to increase the value of therapeutic proteins but also to better understand the role of glycosylation in general and sialylation in particular in fundamental biological processes.Plants are considered an attractive alternative expression platform for therapeutic human glycoproteins because they are cost-effective, highly scalable, free from human pathogens, and, importantly, can carry out complex N-glycosylation (5Stoger E. Ma J.K. Fischer R. Christou P. Curr. Opin. Biotechnol. 2005; 16: 167-173Crossref PubMed Scopus (281) Google Scholar, 6Gleba Y. Klimyuk V. Marillonnet S. Curr. Opin. Biotechnol. 2007; 18: 134-141Crossref PubMed Scopus (307) Google Scholar). In addition, plants glycosylate proteins with considerable uniformity, which may provide an advantage over mammalian-based expression systems. However, plants lack sialylated N-glycans, which restrict their utility as a versatile production platform. Recent studies have shown that the plant N-glycosylation pathway can be modified toward human-type glycosylation (7Cox K.M. Sterling J.D. Regan J.T. Gasdaska J.R. Frantz K.K. Peele C.G. Black A. Passmore D. Moldovan-Loomis C. Srinivasan M. Cuison S. Cardarelli P.M. Dickey L.F. Nat. Biotechnol. 2006; 24: 1591-1597Crossref PubMed Scopus (342) Google Scholar, 8Schuster M. Jost W. Mudde G.C. Wiederkum S. Schwager C. Janzek E. Altmann F. Stadlmann J. Stemmer C. Gorr G. Biotechnol. J. 2007; 2: 700-708Crossref PubMed Scopus (77) Google Scholar, 9Strasser R. Stadlmann J. Schähs M. Stiegler G. Quendler H. Mach L. Glössl J. Weterings K. Pabst M. Steinkellner H. Plant Biotechnol. J. 2008; 6: 392-402Crossref PubMed Scopus (391) Google Scholar). Elimination of plant-specific glycosylation in our laboratory and by others has resulted in the generation of plant lines (e.g. ΔXT/FT) that produce therapeutically relevant proteins carrying a human-like glycosylation profile (GnGn structures). Importantly, monoclonal antibodies produced in such lines exhibited enhanced effector functions (7Cox K.M. Sterling J.D. Regan J.T. Gasdaska J.R. Frantz K.K. Peele C.G. Black A. Passmore D. Moldovan-Loomis C. Srinivasan M. Cuison S. Cardarelli P.M. Dickey L.F. Nat. Biotechnol. 2006; 24: 1591-1597Crossref PubMed Scopus (342) Google Scholar, 8Schuster M. Jost W. Mudde G.C. Wiederkum S. Schwager C. Janzek E. Altmann F. Stadlmann J. Stemmer C. Gorr G. Biotechnol. J. 2007; 2: 700-708Crossref PubMed Scopus (77) Google Scholar, 9Strasser R. Stadlmann J. Schähs M. Stiegler G. Quendler H. Mach L. Glössl J. Weterings K. Pabst M. Steinkellner H. Plant Biotechnol. J. 2008; 6: 392-402Crossref PubMed Scopus (391) Google Scholar). In addition, we demonstrated recently that by overexpressing a modified version of the human β1,4-galactosyltransferase (GalT), plant N-glycans can be extended with β1,4-linked galactose at great uniformity (10Strasser R. Castilho A. Stadlmann J. Kunert R. Quendler H. Gattinger P. Jez J. Rademacher T. Altmann F. Mach L. Steinkellner H. J. Biol. Chem. 2009; 284: 20479-20485Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). Furthermore, these structures serve as acceptor substrates for subsequent terminal sialylation. However, sialylation is particularly difficult to accomplish even in the presence of β1,4-galactosylated structures because plants lack some further essential prerequisites: (i) the biosynthetic capability to produce the sugar nucleotide precursor CMP-sialic acid, specifically CMP-N-acetylneuraminic acid (CMP-Neu5Ac); (ii) a transporter that delivers CMP-sialic acid into the Golgi in sufficient amounts; and (iii) a sialyltransferase (ST) to transfer sialic acid from CMP-Neu5Ac to terminal galactose on the nascent glycoprotein. Moreover, these proteins and their substrates must work in a highly coordinated fashion at different stages of the pathway, and thus organelle-specific targeting of several components is required to enable proper protein sialylation (see Fig. 1). Initial attempts to introduce Neu5Ac residues into plant N-glycans have resulted in the expression of some of these proteins in plants (11Wee E.G. Sherrier D.J. Prime T.A. Dupree P. Plant Cell. 1998; 10: 1759-1768Crossref PubMed Scopus (157) Google Scholar, 12Misaki R. Fujiyama K. Seki T. Biochem. Biophys. Res. Commun. 2006; 339: 1184-1189Crossref PubMed Scopus (39) Google Scholar), and recent progress in our laboratory allowed the synthesis of the sugar nucleotide CMP-Neu5Ac from endogenous metabolites by the simultaneous overexpression of three mammalian enzymes in Arabidopsis thaliana (13Castilho A. Pabst M. Leonard R. Veit C. Altmann F. Mach L. Glössl J. Strasser R. Steinkellner H. Plant Physiol. 2008; 147: 331-339Crossref PubMed Scopus (62) Google Scholar). However, in planta protein sialylation has not yet been achieved.In this study, we aimed to achieve in planta sialylation in Nicotiana benthamiana, a tobacco-related plant species particularly well suited for recombinant protein expression (14Giritch A. Marillonnet S. Engler C. van Eldik G. Botterman J. Klimyuk V. Gleba Y. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 14701-14706Crossref PubMed Scopus (344) Google Scholar). Six mammalian proteins comprising the sialic acid pathway were co-expressed together with a monoclonal antibody (mAb) in N. benthamiana wild type (WT) and glycosylation mutant plants thereof. Purified mAbs were subjected to glycosylation analyses and tested for functional integrity.DISCUSSIONHere we report the efficient in planta sialylation of a therapeutic protein as exemplified by a recombinant mAb. This was achieved by the coordinated overexpression of six mammalian proteins that act at various stages of the biosynthetic pathway in different subcellular compartments. Quantitative sialylation was facilitated by the specific modification of some of these proteins, i.e. elimination of activity-destroying amino acid sequences (in the case of CMAS) (13Castilho A. Pabst M. Leonard R. Veit C. Altmann F. Mach L. Glössl J. Strasser R. Steinkellner H. Plant Physiol. 2008; 147: 331-339Crossref PubMed Scopus (62) Google Scholar) and by enabling proper subcellular localization (in the case of ST-GalT) (10Strasser R. Castilho A. Stadlmann J. Kunert R. Quendler H. Gattinger P. Jez J. Rademacher T. Altmann F. Mach L. Steinkellner H. J. Biol. Chem. 2009; 284: 20479-20485Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). Notably, nearly all of the available acceptor substrates (i.e. galactosylated glycans) present in WT and in the glycosylation mutants were sialylated. The synthesis of incompletely processed structures such as MNai, MNaFi, and MNaXi is most likely due to inappropriate overexpression of ST-GalT interfering with the action of GlcNAc-T II. Incompletely processed glycans with one antenna terminating in mannose (MAXFi and MAi, Fig. 6a and data not shown, respectively) were present in WT and ΔXT/FT plants expressing only ST-GalT, an observation already made previously (10Strasser R. Castilho A. Stadlmann J. Kunert R. Quendler H. Gattinger P. Jez J. Rademacher T. Altmann F. Mach L. Steinkellner H. J. Biol. Chem. 2009; 284: 20479-20485Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). This shortcoming may be overcome by optimizing the expression system as demonstrated for stably transformed GalT+ plants (10Strasser R. Castilho A. Stadlmann J. Kunert R. Quendler H. Gattinger P. Jez J. Rademacher T. Altmann F. Mach L. Steinkellner H. J. Biol. Chem. 2009; 284: 20479-20485Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). Note that GalT+ plants were not considered for sialylation experiments because they are not yet in a homozygous stage.The high amount of sialylated glycoforms of 2G12 upon expressing proteins of the mammalian biosynthetic pathway points at a considerable degree of functional conservation between plant and mammalian cells. Moreover, the entire procedure did not interfere with IgG expression, which is remarkable in the light of the complexity of the process.As reported for A. thaliana transformed with GNE, NANS, and CMAS (13Castilho A. Pabst M. Leonard R. Veit C. Altmann F. Mach L. Glössl J. Strasser R. Steinkellner H. Plant Physiol. 2008; 147: 331-339Crossref PubMed Scopus (62) Google Scholar), transient expression of these genes in N. benthamiana efficiently converted ManNAc-6-phosphate to Neu5Ac rather than to Neu5Ac-9-phosphate, suggesting the presence of a so far uncharacterized endogenous phosphatase acting on sialic acid-9-phosphate. On the other hand, although the presence of plant homologues for the CMP-Neu5Ac transporter and sialyltransferases has been suggested (24Bakker H. Routier F. Ashikov A. Neumann D. Bosch D. Gerardy-Schahn R. Carbohydr. Res. 2008; 343: 2148-2152Crossref PubMed Scopus (38) Google Scholar, 25Takashima S. Abe T. Yoshida S. Kawahigashi H. Saito T. Tsuji S. Tsujimoto M. J. Biochem. 2006; 139: 279-287Crossref PubMed Scopus (29) Google Scholar), our experiments indicate that all required mammalian genes need to be expressed to achieve in planta protein sialylation. Notably, transient sialylation did not result in any obvious phenotype in the infiltrated leaves, indicating that at least certain plant tissues/organs can tolerate the incorporation of highly charged sialic acid into proteins. This is not surprising because N-glycans of total plant proteins derived from plants expressing all six genes of the sialylation pathway exhibited a relatively low amount of sialic acid (supplemental Fig. 1), in contrast to the efficient sialylation of the secreted mAb. The formation of disialylated mAb is remarkable because in human B-lymphocytes, α2,6-ST preferentially generates monosialylated Fc glycans (26Barb A.W. Brady E.K. Prestegard J.H. Biochemistry. 2009; 48: 9705-9707Crossref PubMed Scopus (65) Google Scholar). In this light, the in planta sialylation of a recombinant protein is extremely high and significantly outperforms the sialylation ability of commonly used mammalian cell-based expression lines that generate low amounts of sialylated N-glycans (2Stadlmann J. Pabst M. Kolarich D. Kunert R. Altmann F. Proteomics. 2008; 8: 2858-2871Crossref PubMed Scopus (262) Google Scholar). The rarely used Nawalma cell line is an exception, generating mAbs with an increased sialylated Fc domain (27Stadlmann J. Weber A. Pabst M. Anderle H. Kunert R. Ehrlich H.J. Schwarz P.H. Altmann F. Proteomics. 2009; 9: 4143-4153Crossref PubMed Scopus (86) Google Scholar). A substrate with more exposed N-glycans than Fcs would be expected to be furnished with higher amounts of disialylated N-glycans. This glycoform has indeed been generated for erythropoietin and lactotransferrin upon expression in glyco-engineered Pichia pastoris (28Hamilton S.R. Davidson R.C. Sethuraman N. Nett J.H. Jiang Y. Rios S. Bobrowicz P. Stadheim T.A. Li H. Choi B.K. Hopkins D. Wischnewski H. Roser J. Mitchell T. Strawbridge R.R. Hoopes J. Wildt S. Gerngross T.U. Science. 2006; 313: 1441-1443Crossref PubMed Scopus (409) Google Scholar). The sialylation of lactotransferrin was performed in vitro for reasons that were not disclosed (29Choi B.K. Actor J.K. Rios S. d'Anjou M. Stadheim T.A. Warburton S. Giaccone E. Cukan M. Li H. Kull A. Sharkey N. Gollnick P. Kocieba M. Artym J. Zimecki M. Kruzel M.L. Wildt S. Glycoconj. J. 2008; 25: 581-593Crossref PubMed Scopus (55) Google Scholar).Here we show that sialylated 2G12 exhibits similar in vitro HIV neutralization potency to other glycoforms derived from plants and CHO cells, demonstrating full integrity of the protein. Notably, this neutralization experiment entirely relies on antigen-antibody binding and does not involve effector functions of the antibodies. Several studies have demonstrated the impact of proper Fc glycosylation for the biological activities of mAbs with the general agreement that mAbs lacking core α1,6-fucose exhibit a dramatically increased antibody-dependent cell mediated cytotoxicity activity (30Jefferis R. Nat. Rev. Drug Discov. 2009; 8: 226-234Crossref PubMed Scopus (646) Google Scholar, 31Raju T.S. Curr. Opin. Immunol. 2008; 20: 471-478Crossref PubMed Scopus (419) Google Scholar). In contrast, increased sialylation of IgGs results in reduced antibody-dependent cell mediated cytotoxicity activity because of a decreased binding to the FcγRIIIa receptor (4Scallon B.J. Tam S.H. McCarthy S.G. Cai A.N. Raju T.S. Mol. Immunol. 2007; 44: 1524-1534Crossref PubMed Scopus (317) Google Scholar, 32Kaneko Y. Nimmerjahn F. Ravetch J.V. Science. 2006; 313: 670-673Crossref PubMed Scopus (1352) Google Scholar). On the other hand, Fc sialylation of serum IgG is a major determinant in anti-inflammatory processes in autoimmune disorders (3Anthony R.M. Nimmerjahn F. Ashline D.J. Reinhold V.N. Paulson J.C. Ravetch J.V. Science. 2008; 320: 373-376Crossref PubMed Scopus (637) Google Scholar). The rapid generation of highly α2,6-sialylated Fc as described in this work may substantially contribute to further elucidation of the underlying molecular mechanisms and may facilitate the generation of hypersialylated IgGs for the use as potent drugs to treat autoimmune diseases.This proof of concept study completes many years of intensive efforts to fully humanize plant glycosylation. Moreover, it is the first report demonstrating that a multicellular organism naturally lacking sialylated glycoconjugates can incorporate the entire biosynthetic pathway for protein sialylation. The lack of any phenotype in infiltrated leaves indicates the feasibility of generating plants stably expressing the entire pathway. Recent advances in multigene delivery (33Dafny-Yelin M. Tzfira T. Plant Physiol. 2007; 145: 1118-1128Crossref PubMed Scopus (104) Google Scholar) should allow the efficient generation of such plants to accomplish in planta sialylation in the future. The availability of such lines, together with recently generated glyco-engineered mutants displaying human-type glycosylation of great uniformity (9Strasser R. Stadlmann J. Schähs M. Stiegler G. Quendler H. Mach L. Glössl J. Weterings K. Pabst M. Steinkellner H. Plant Biotechnol. J. 2008; 6: 392-402Crossref PubMed Scopus (391) Google Scholar, 10Strasser R. Castilho A. Stadlmann J. Kunert R. Quendler H. Gattinger P. Jez J. Rademacher T. Altmann F. Mach L. Steinkellner H. J. Biol. Chem. 2009; 284: 20479-20485Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar), pave the way not only for generating therapeutic glycoproteins with optimized biological activities obtained by a customized N-glycosylation profile but also for studying the impact of different glycoforms in biological processes. In case whole plants would not tolerate protein sialylation during their complete developmental process, one of the six genes could be transiently expressed together with the protein of interest. Another alternative to this hurdle would be the use of tissue-specific or inducible promoters to specifically turn on protein sialylation in leaves.Glycoengineered plants in combination with newly developed plant virus-based transient expression systems, allowing the generation of a virtually unlimited number of different proteins at high amounts within 1 week after DNA construct delivery (6Gleba Y. Klimyuk V. Marillonnet S. Curr. Opin. Biotechnol. 2007; 18: 134-141Crossref PubMed Scopus (307) Google Scholar, 34Sainsbury F. Lomonossoff G.P. Plant Physiol. 2008; 148: 1212-1218Crossref PubMed Scopus (228) Google Scholar), provide a considerable advantage over existing glyco-modified expression platforms, including yeast and mammalian cells. Besides the importance of this work in biotechnological applications, it may serve as a model for the manipulation of complex metabolic pathways into plants for the generation of varieties with new traits. IntroductionThe outstanding specificity of therapeutic glycoproteins places them among the fastest growing class of pharmaceutical products. Many of these drugs need terminal sialylation, the final and most complex step of human N-glycosylation, for optimal therapeutic potency. Therefore manufacturing is currently restricted to mammalian cell-based systems that are able to perform this important posttranslational modification, although with major limitations. The naturally present glycosylation repertoire of mammalian host cells promotes the generation of a number of different terminal structures and thus leads to heterogeneous glycosylation. Moreover, these modifications may differ from the authentic human glycosylation. For example, CHO 4The abbreviations used are: CHOChinese hamster ovaryGalTβ1,4-galactosyltransferaseST-GalTmodified β1,4-galactosyltransferaseSTα2,6-sialyltransferaseCMASCMP-N-acetylneuraminic acid synthetaseNANSN-acetylneuraminic acid phosphate synthaseGNEUDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinaseCSTCMP-Neu5Ac transportermAbmonoclonal antibodyHIVhuman immunodeficiency virusGFPgreen fluorescent proteinRubiscoribulose-bisphosphate carboxylase/oxygenaseWTwild typeMES4-morpholineethanesulfonic acidMSmass spectrometryMS/MStandem MSLCliquid chromatographyESIelectrospray mass ionizationTOFtime-of-flight. cells, widely used for the expression of recombinant glycoproteins, do not naturally produce human type α2,6-sialylation. These shortcomings in many cases lead to drugs with reduced biological potency, of which recombinant hormones such as human erythropoietin and interferons are prominent examples (1Bork K. Horstkorte R. Weidemann W. J. Pharm. Sci. 2009; 98: 3499-3508Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). In addition, about 10% of the N-glycans in the Fc region of human serum IgG is sialylated (2Stadlmann J. Pabst M. Kolarich D. Kunert R. Altmann F. Proteomics. 2008; 8: 2858-2871Crossref PubMed Scopus (262) Google Scholar). Although the anti-inflammatory effects of sialylated IgG have recently been demonstrated (3Anthony R.M. Nimmerjahn F. Ashline D.J. Reinhold V.N. Paulson J.C. Ravetch J.V. Science. 2008; 320: 373-376Crossref PubMed Scopus (637) Google Scholar), very little information on the impact of this common modification at the molecular level is available (4Scallon B.J. Tam S.H. McCarthy S.G. Cai A.N. Raju T.S. Mol. Immunol. 2007; 44: 1524-1534Crossref PubMed Scopus (317) Google Scholar). This explains the general interest in novel expression hosts and strategies to engineer glycosylation not only to increase the value of therapeutic proteins but also to better understand the role of glycosylation in general and sialylation in particular in fundamental biological processes.Plants are considered an attractive alternative expression platform for therapeutic human glycoproteins because they are cost-effective, highly scalable, free from human pathogens, and, importantly, can carry out complex N-glycosylation (5Stoger E. Ma J.K. Fischer R. Christou P. Curr. Opin. Biotechnol. 2005; 16: 167-173Crossref PubMed Scopus (281) Google Scholar, 6Gleba Y. Klimyuk V. Marillonnet S. Curr. Opin. Biotechnol. 2007; 18: 134-141Crossref PubMed Scopus (307) Google Scholar). In addition, plants glycosylate proteins with considerable uniformity, which may provide an advantage over mammalian-based expression systems. However, plants lack sialylated N-glycans, which restrict their utility as a versatile production platform. Recent studies have shown that the plant N-glycosylation pathway can be modified toward human-type glycosylation (7Cox K.M. Sterling J.D. Regan J.T. Gasdaska J.R. Frantz K.K. Peele C.G. Black A. Passmore D. Moldovan-Loomis C. Srinivasan M. Cuison S. Cardarelli P.M. Dickey L.F. Nat. Biotechnol. 2006; 24: 1591-1597Crossref PubMed Scopus (342) Google Scholar, 8Schuster M. Jost W. Mudde G.C. Wiederkum S. Schwager C. Janzek E. Altmann F. Stadlmann J. Stemmer C. Gorr G. Biotechnol. J. 2007; 2: 700-708Crossref PubMed Scopus (77) Google Scholar, 9Strasser R. Stadlmann J. Schähs M. Stiegler G. Quendler H. Mach L. Glössl J. Weterings K. Pabst M. Steinkellner H. Plant Biotechnol. J. 2008; 6: 392-402Crossref PubMed Scopus (391) Google Scholar). Elimination of plant-specific glycosylation in our laboratory and by others has resulted in the generation of plant lines (e.g. ΔXT/FT) that produce therapeutically relevant proteins carrying a human-like glycosylation profile (GnGn structures). Importantly, monoclonal antibodies produced in such lines exhibited enhanced effector functions (7Cox K.M. Sterling J.D. Regan J.T. Gasdaska J.R. Frantz K.K. Peele C.G. Black A. Passmore D. Moldovan-Loomis C. Srinivasan M. Cuison S. Cardarelli P.M. Dickey L.F. Nat. Biotechnol. 2006; 24: 1591-1597Crossref PubMed Scopus (342) Google Scholar, 8Schuster M. Jost W. Mudde G.C. Wiederkum S. Schwager C. Janzek E. Altmann F. Stadlmann J. Stemmer C. Gorr G. Biotechnol. J. 2007; 2: 700-708Crossref PubMed Scopus (77) Google Scholar, 9Strasser R. Stadlmann J. Schähs M. Stiegler G. Quendler H. Mach L. Glössl J. Weterings K. Pabst M. Steinkellner H. Plant Biotechnol. J. 2008; 6: 392-402Crossref PubMed Scopus (391) Google Scholar). In addition, we demonstrated recently that by overexpressing a modified version of the human β1,4-galactosyltransferase (GalT), plant N-glycans can be extended with β1,4-linked galactose at great uniformity (10Strasser R. Castilho A. Stadlmann J. Kunert R. Quendler H. Gattinger P. Jez J. Rademacher T. Altmann F. Mach L. Steinkellner H. J. Biol. Chem. 2009; 284: 20479-20485Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). Furthermore, these structures serve as acceptor substrates for subsequent terminal sialylation. However, sialylation is particularly difficult to accomplish even in the presence of β1,4-galactosylated structures because plants lack some further essential prerequisites: (i) the biosynthetic capability to produce the sugar nucleotide precursor CMP-sialic acid, specifically CMP-N-acetylneuraminic acid (CMP-Neu5Ac); (ii) a transporter that delivers CMP-sialic acid into the Golgi in sufficient amounts; and (iii) a sialyltransferase (ST) to transfer sialic acid from CMP-Neu5Ac to terminal galactose on the nascent glycoprotein. Moreover, these proteins and their substrates must work in a highly coordinated fashion at different stages of the pathway, and thus organelle-specific targeting of several components is required to enable proper protein sialylation (see Fig. 1). Initial attempts to introduce Neu5Ac residues into plant N-glycans have resulted in the expression of some of these proteins in plants (11Wee E.G. Sherrier D.J. Prime T.A. Dupree P. Plant Cell. 1998; 10: 1759-1768Crossref PubMed Scopus (157) Google Scholar, 12Misaki R. Fujiyama K. Seki T. Biochem. Biophys. Res. Commun. 2006; 339: 1184-1189Crossref PubMed Scopus (39) Google Scholar), and recent progress in our laboratory allowed the synthesis of the sugar nucleotide CMP-Neu5Ac from endogenous metabolites by the simultaneous overexpression of three mammalian enzymes in Arabidopsis thaliana (13Castilho A. Pabst M. Leonard R. Veit C. Altmann F. Mach L. Glössl J. Strasser R. Steinkellner H. Plant Physiol. 2008; 147: 331-339Crossref PubMed Scopus (62) Google Scholar). However, in planta protein sialylation has not yet been achieved.In this study, we aimed to achieve in planta sialylation in Nicotiana benthamiana, a tobacco-related plant species particularly well suited for recombinant protein expression (14Giritch A. Marillonnet S. Engler C. van Eldik G. Botterman J. Klimyuk V. Gleba Y. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 14701-14706Crossref PubMed Scopus (344) Google Scholar). Six mammalian proteins comprising the sialic acid pathway were co-expressed together with a monoclonal antibody (mAb) in N. benthamiana wild type (WT) and glycosylation mutant plants thereof. Purified mAbs were subjected to glycosylation analyses and tested for functional integrity." @default.
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• W2026760618 date "2010-05-01" @default.
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• W2026760618 title "In Planta Protein Sialylation through Overexpression of the Respective Mammalian Pathway" @default.
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