Hydroponic Treatment of Nicotiana benthamiana with Kifunensine Modifies the N-glycans of Recombinant Glycoprotein Antigens to Predominantly Man9 High-Mannose Type upon Transient Overexpression.

Sugata Roychowdhury,Kazuhito Fujiyama,Hiroyuki Kajiura,Krystal T Hamorsky,Young J Oh,Nobuyuki Matoba

doi:10.3389/fpls.2018.00062

Abstract

Nicotiana benthamiana transient overexpression systems offer unique advantages for rapid and scalable biopharmaceuticals production, including high scalability and eukaryotic post-translational modifications such as N-glycosylation. High-mannose-type glycans (HMGs) of glycoprotein antigens have been implicated in the effectiveness of some subunit vaccines. In particular, Man9GlcNAc2 (Man9) has high binding affinity to mannose-specific C-type lectin receptors such as the mannose receptor and dendritic cell-specific intracellular adhesion molecule 3-grabbing non-integrin (DC-SIGN). Here, we investigated the effect of kifunensine, an α-mannosidase I inhibitor, supplemented in a hydroponic culture of N. benthamiana for the production of Man9-rich HMG glycoproteins, using N-glycosylated cholera toxin B subunit (gCTB) and human immunodeficiency virus gp120 that are tagged with a H/KDEL endoplasmic reticulum retention signal as model vaccine antigens. Biochemical analysis using anti-fucose and anti-xylose antibodies as well as Endo H and PNGase F digestion showed that kifunensine treatment effectively reduced plant-specific glycoforms while increasing HMGs in the N-glycan compositions of gCTB. Detailed glycan profiling revealed that plant-produced gp120 had a glycan profile bearing mostly HMGs regardless of kifunensine treatment. However, the gp120 produced under kifunensine-treatment conditions showed Man9 being the most prominent glycoform (64.5%), while the protein produced without kifunensine had a substantially lower Man9 composition (20.3%). Our results open up possibilities for efficient production of highly mannosylated recombinant vaccine antigens in plants.

Highlights

Over the past decade, Nicotiana benthamiana expression systems using viral and non-viral vectors have become viable platforms for the production of recombinant proteins (Matoba et al, 2011; Whaley et al, 2011; Chen et al, 2013; Nandi et al, 2016)
For transient overexpression of glycosylated cholera toxin B subunit (gCTB) in N. benthamiana, the magnICON tobamovirus replicon vector was employed, which was delivered via vacuum-mediated agroinfiltration (Hamorsky et al, 2015)
This is due to incomplete N-glycan occupancy at Asn4 of the protein; the upper band corresponds to glycosylated gCTB and the lower band indicates the aglycosylated form (Hamorsky et al, 2015)

Summary

Introduction

Nicotiana benthamiana expression systems using viral and non-viral vectors have become viable platforms for the production of recombinant proteins (Matoba et al, 2011; Whaley et al, 2011; Chen et al, 2013; Nandi et al, 2016). Our results in the present work demonstrate that recombinant glycoproteins transiently expressed in N. benthamiana have predominantly Man9 HMGs upon hydroponically treating the plant with kifunensine after vector inoculation, providing a new method for the efficient production of highly mannosylated antigens for vaccine development.

Results

Conclusion