Research

Function and regulation of plant aquaporins

Aquaporins (AQPs) are channels facilitating the movement of water and small solutes across cellular membranes. Plants appear to express a surprisingly high number of AQP homologues. On the basis of sequence comparison, higher plant AQPs are classified into five subfamilies, the plasma membrane intrinsic proteins (PIPs), the tonoplast intrinsic protein (TIP), the NOD26-like intrinsic protein (NIP), the small basic intrinsic proteins (SIPs) and the uncharacterized X intrinsic proteins (XIPs). AQPs are thought to be involved in the regulation of transcellular water flow for long-distance transport in the root and leaf tissues. Their role is also critical for short-distance water transport and osmotic adjustments within a cell and between the cytoplasm and the cell wall space. However, some AQP isoforms play important roles in many other processes, such as gas and nutrient uptake and translocation, metalloid homeostasis, signal transduction. AQP abundance is regulated developmentally in a cell-specific manner and by environmental signals. The activity of AQPs is also regulated by different post-translational regulation mechanisms, which provide an efficient way for rapid and reversible regulation of the water membrane permeability.

The research project aims at understanding the function and regulation of plant AQPs at the cellular level and in the whole plant subjected to various environmental conditions.

 

Plant Molecular Farming: Production of pharmaceutical proteins in plant cell suspension

Nicotiana tabacum BY-2 suspension cellsPlant cell suspensions represent an interesting production platform for therapeutic glycoproteins, especially because of the absence of contamination by human pathogens, the straightforward implementable GMP-compatible culture conditions and the presence of high eukaryote post-translational modifications like N-glycosylation. In that context, the Nicotiana tabacum Bright Yellow 2 (BY-2) line is one of the most commonly used plant cell lines for producing biopharmaceutical glycoproteins because it grows fast and it is easily genetically transformed.

This platform is still under development and several strategies are being explored to exploit its full-fledged potential. Our team has already produced pharmacological proteins in plants and BY-2 plant cells: this comprises several antibodies and vaccines (cytomegalovirus (CMV), influenza virus, Lyme disease). Our know-how includes constitutive and inducible transcription promoters characterization, inactivation of gene expression using RNA interference or genome editing by CRISPR/Cas9, culture medium optimization, protein purification, identification of proteases and modification of the glycosylation machinery by genetic engineering.

Our investigations focus on both quantitative and qualitative aspects of pharmaceutical protein production in BY-2 cell culture.

A first project aims to increase further the productivity of our BY-2 cell lines. A wide range of cis-regulatory elements (including promoters, terminators, 5’ and 3’ untranslated regions and scaffold attachment regions) can be combined and evaluated in vivo. In addition, the impact of RNA silencing process in BY-2 cells on the expression of transgene is currently under investigation

In parallel, we are developing a plant cell platform for the production of vaccine glycoproteins with homogeneous and pre-defined N-glycosylation profiles. Indeed, N-glycosylation is a key post-translational modification, which deeply influences the properties of glycoproteins, e.g., making them more soluble, protecting them from proteolysis and covering antigenic sites. More particularly, this project consists of obtaining several BY-2 cell lines modified in their N-glycosylation pathway by different genetic engineering approaches (knock out of endogenous genes and expression of ectopic genes). These cell lines are being evaluated by monitoring the expression and the N-glycosylation profile of a vaccine candidate from CMV.