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.
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-α-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 β-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.


CONCLUSIONS
Plant bioreactors can be engineered that are capable of producing Tn antigen-containing recombinant therapeutics.
Daskalova
Sasha M
SM
Center for Infectious Diseases and Vaccinology, The Biodesign Institute, Arizona State University, Tempe, AZ 85287, USA. sashamv@gmail.com
Radder
Josiah E
JE
Cichacz
Zbigniew A
ZA
Olsen
Sam H
SH
Tsaprailis
George
G
Mason
Hugh
H
Lopez
Linda C
LC
D015295:Antigens, Tumor-Associated, Carbohydrate / Q000096:biosynthesis* / Q000235:genetics; D002238:Carbohydrate Epimerases / Q000235:genetics / Q000378:metabolism; D005818:Genetic Engineering / Q000379:methods; D006031:Glycosylation; D006801:Humans; D018396:Mucin-1 / Q000737:chemistry*; D017350:N-Acetylgalactosaminyltransferases / Q000235:genetics / Q000378:metabolism*; D030821:Plants, Genetically Modified / Q000235:genetics / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D018749:RNA, Plant / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D014026:Nicotiana / Q000235:genetics* / Q000378:metabolism; D014170:Transformation, Genetic; D000097763:Polypeptide N-acetylgalactosaminyltransferase
D015295:Antigens, Tumor-Associated, Carbohydrate; D002238:Carbohydrate Epimerases; D018396:Mucin-1; D017350:N-Acetylgalactosaminyltransferases; D018749:RNA, Plant; D011993:Recombinant Fusion Proteins; D000097763:Polypeptide N-acetylgalactosaminyltransferase; C040286:Tn antigen; C023223:UDP-N-acetylglucosamine 4-epimerase