Modifying N-linked glycosylation in tobacco to produce human-type post-translational modifications

Plant cells are a capable system for producing high value proteins for a variety of applications, and challenge popular host organisms such as mammalian cells, bacteria or yeasts. However, plants do not perform post translation protein modification in the same manner as mammalian cells, most critically in terms of N-linked glycosylation. This can impact both protein functionality and stability, key for human therapeutics intended for production in plants. This obstacle can be approached by creating a plant-based system capable of “humanizing” proteins of interest in terms of N-linked glycosylation, resulting in plant-produced proteins with a glycan profile as would occur in a mammalian host system, making them more suitable for therapeutic applications. For this, four human glycosylation enzymes (HuGEs) involved in N-linked glycosylation N- acetylglucosaminyltransferase IV and V (GNTIV and GNTV), β-1,4-galactosyltransferase (B4GALT1), and α-2,6-sialyltransferase (ST6GAL) need to be expressed in plant cells. As glycosylation is a stepwise process, enzymes need to localise to either medial or trans-Golgi body cisternae. For this, a protein targeting strategy of replacing the mammalian N-terminal Golgi targeting domains on HuGEs (Cytoplasmic-Transmembrane-Stem (CTS) regions) with plant–specific domains, MUR3 or FUT13, were tested transiently and were successful in redirecting localisation. This was conducted via high-resolution dynamic confocal microscopy using an analysis pipeline based upon distance between peak fluorescence intensity, relative to known Golgi markers MNS1 and ST. This analysis demonstrated that the modified MUR3-GNTIV and MUR3-GNTV were successfully targeted to the medial-Golgi cisternae while FUT13-ST6GAL and FUT13-B4GALT1 were targeted to trans-Golgi cisternae. Following successful expression and targeting of the four HuGEs transiently, these four enzymes were combined into two expression cassettes for stable plant transformation. These consisted of a N-terminal CTS domain, the catalytic subunit of the first enzyme, a linker domain, and the second enzyme catalytic subunit without attached fluorescent protein. This halved the insert number necessary to generate stable glycomodified lines, transformed using the modified HuGEs in wild type and plant-glycan deficient (ΔXFT) lines of N. benthamiana. Following growth, resulting seedlings of this line were successfully screened for presence of each of the four HuGE catalytic domains by RT-PCR against a 300BP sequence specific to each enzyme. Human proteins of interest LAL, IgE and IgG were then transiently expressed in these glycomodified lines and lectin binding assays performed against the plant extracts, either dot blot or by SDS-PAGE. The results of the lectin binding assays potentially indicate altered glycomodification in the HuGE stable lines but are ultimately inconclusive.

Grantor dc:publisher

Oxford Brookes University

Author dc:creator

• McGinness, Alastair

Contributors dc:contributor

• Kriechbaumer, Verena
• Brooks, Susan
• Strasser, Richard

Statement dc:rights

• All rights reserved

Language dc:language

en

DOI dc:identifier

https://doi.org/10.24384/r9y3-xh28

OAI identifier oai:identifier

tle:69538e08-544d-4278-8086-45c5d33a3f82:d6bd9758-527a-46cd-bfe2-c433766e8fca:1