Engineering of N. benthamiana L. plants for production of N-acetylgalactosamine-glycosylated proteins - towards development of a plant-based platform for production of protein therapeutics with mucin type O-glycosylation

Persistent Link:
http://hdl.handle.net/10150/610340
Title:
Engineering of N. benthamiana L. plants for production of N-acetylgalactosamine-glycosylated proteins - towards development of a plant-based platform for production of protein therapeutics with mucin type O-glycosylation
Author:
Daskalova, Sasha; Radder, Josiah; Cichacz, Zbigniew; Olsen, Sam; Tsaprailis, George; Mason, Hugh; Lopez, Linda
Affiliation:
Center for Infectious Diseases and Vaccinology, The Biodesign Institute, Arizona State University, Tempe, AZ 85287, USA; Center for Innovations in Medicine, The Biodesign Institute, Arizona State University, Tempe, AZ 85287, USA; Center for Toxicology, University of Arizona, Tucson, AZ 85721, USA
Issue Date:
2010
Publisher:
BioMed Central
Citation:
Daskalova et al. BMC Biotechnology 2010, 10:62 http://www.biomedcentral.com/1472-6750/10/62
Journal:
BMC Biotechnology
Rights:
© 2010 Daskalova et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0)
Collection Information:
This item is part of the UA Faculty Publications collection. For more information this item or other items in the UA Campus Repository, contact the University of Arizona Libraries at repository@u.library.arizona.edu.
Abstract:
BACKGROUND:Mucin type O-glycosylation is one of the most common types of post-translational modifications that impacts stability and biological functions of many mammalian proteins. A large family of UDP-GalNAc polypeptide:N-acetyl-alpha-galactosaminyltransferases (GalNAc-Ts) catalyzes the first step of mucin type O-glycosylation by transferring GalNAc to serine and/or threonine residues of acceptor polypeptides. Plants do not have the enzyme machinery to perform this process, thus restricting their use as bioreactors for production of recombinant therapeutic proteins.RESULTS:The present study demonstrates that an isoform of the human GalNAc-Ts family, GalNAc-T2, retains its localization and functionality upon expression in N. benthamiana L. plants. The recombinant enzyme resides in the Golgi as evidenced by the fluorescence distribution pattern of the GalNAc-T2:GFP fusion and alteration of the fluorescence signature upon treatment with Brefeldin A. A GalNAc-T2-specific acceptor peptide, the 113-136 aa fragment of chorionic gonadotropin beta-subunit, is glycosylated in vitro by the plant-produced enzyme at the "native" GalNAc attachment sites, Ser-121 and Ser-127. Ectopic expression of GalNAc-T2 is sufficient to "arm" tobacco cells with the ability to perform GalNAc-glycosylation, as evidenced by the attachment of GalNAc to Thr-119 of the endogenous enzyme endochitinase. However, glycosylation of highly expressed recombinant glycoproteins, like magnICON-expressed E. coli enterotoxin B subunit:H. sapiens mucin 1 tandem repeat-derived peptide fusion protein (LTBMUC1), is limited by the low endogenous UDP-GalNAc substrate pool and the insufficient translocation of UDP-GalNAc to the Golgi lumen. Further genetic engineering of the GalNAc-T2 plants by co-expressing Y. enterocolitica UDP-GlcNAc 4-epimerase gene and C. elegans UDP-GlcNAc/UDP-GalNAc transporter gene overcomes these limitations as indicated by the expression of the model LTBMUC1 protein exclusively as a glycoform.CONCLUSION:Plant bioreactors can be engineered that are capable of producing Tn antigen-containing recombinant therapeutics.
EISSN:
1472-6750
DOI:
10.1186/1472-6750-10-62
Version:
Final published version
Additional Links:
http://www.biomedcentral.com/1472-6750/10/62

Full metadata record

DC FieldValue Language
dc.contributor.authorDaskalova, Sashaen
dc.contributor.authorRadder, Josiahen
dc.contributor.authorCichacz, Zbigniewen
dc.contributor.authorOlsen, Samen
dc.contributor.authorTsaprailis, Georgeen
dc.contributor.authorMason, Hughen
dc.contributor.authorLopez, Lindaen
dc.date.accessioned2016-05-20T09:04:39Z-
dc.date.available2016-05-20T09:04:39Z-
dc.date.issued2010en
dc.identifier.citationDaskalova et al. BMC Biotechnology 2010, 10:62 http://www.biomedcentral.com/1472-6750/10/62en
dc.identifier.doi10.1186/1472-6750-10-62en
dc.identifier.urihttp://hdl.handle.net/10150/610340-
dc.description.abstractBACKGROUND:Mucin type O-glycosylation is one of the most common types of post-translational modifications that impacts stability and biological functions of many mammalian proteins. A large family of UDP-GalNAc polypeptide:N-acetyl-alpha-galactosaminyltransferases (GalNAc-Ts) catalyzes the first step of mucin type O-glycosylation by transferring GalNAc to serine and/or threonine residues of acceptor polypeptides. Plants do not have the enzyme machinery to perform this process, thus restricting their use as bioreactors for production of recombinant therapeutic proteins.RESULTS:The present study demonstrates that an isoform of the human GalNAc-Ts family, GalNAc-T2, retains its localization and functionality upon expression in N. benthamiana L. plants. The recombinant enzyme resides in the Golgi as evidenced by the fluorescence distribution pattern of the GalNAc-T2:GFP fusion and alteration of the fluorescence signature upon treatment with Brefeldin A. A GalNAc-T2-specific acceptor peptide, the 113-136 aa fragment of chorionic gonadotropin beta-subunit, is glycosylated in vitro by the plant-produced enzyme at the "native" GalNAc attachment sites, Ser-121 and Ser-127. Ectopic expression of GalNAc-T2 is sufficient to "arm" tobacco cells with the ability to perform GalNAc-glycosylation, as evidenced by the attachment of GalNAc to Thr-119 of the endogenous enzyme endochitinase. However, glycosylation of highly expressed recombinant glycoproteins, like magnICON-expressed E. coli enterotoxin B subunit:H. sapiens mucin 1 tandem repeat-derived peptide fusion protein (LTBMUC1), is limited by the low endogenous UDP-GalNAc substrate pool and the insufficient translocation of UDP-GalNAc to the Golgi lumen. Further genetic engineering of the GalNAc-T2 plants by co-expressing Y. enterocolitica UDP-GlcNAc 4-epimerase gene and C. elegans UDP-GlcNAc/UDP-GalNAc transporter gene overcomes these limitations as indicated by the expression of the model LTBMUC1 protein exclusively as a glycoform.CONCLUSION:Plant bioreactors can be engineered that are capable of producing Tn antigen-containing recombinant therapeutics.en
dc.language.isoenen
dc.publisherBioMed Centralen
dc.relation.urlhttp://www.biomedcentral.com/1472-6750/10/62en
dc.rights© 2010 Daskalova et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0)en
dc.titleEngineering of N. benthamiana L. plants for production of N-acetylgalactosamine-glycosylated proteins - towards development of a plant-based platform for production of protein therapeutics with mucin type O-glycosylationen
dc.typeArticleen
dc.identifier.eissn1472-6750en
dc.contributor.departmentCenter for Infectious Diseases and Vaccinology, The Biodesign Institute, Arizona State University, Tempe, AZ 85287, USAen
dc.contributor.departmentCenter for Innovations in Medicine, The Biodesign Institute, Arizona State University, Tempe, AZ 85287, USAen
dc.contributor.departmentCenter for Toxicology, University of Arizona, Tucson, AZ 85721, USAen
dc.identifier.journalBMC Biotechnologyen
dc.description.collectioninformationThis item is part of the UA Faculty Publications collection. For more information this item or other items in the UA Campus Repository, contact the University of Arizona Libraries at repository@u.library.arizona.edu.en
dc.eprint.versionFinal published versionen
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