Research

In all eukaryotic cells, the endoplasmic reticulum (ER) has a central role in protein biosynthesis and maturation. Folding of newly synthesized secretory and membrane proteins takes place in the lumen of the ER. Properly folded and assembled proteins exit the ER and continue their journey in the secretory pathway. However, protein folding is error-prone and the accumulation of misfolded or surplus proteins endangers the cellular homeostasis and subsequently the survival of organisms especially under adverse environmental conditions. Consequently, cells have established sophisticated quality control processes that ensure the export of biologically active proteins and the elimination of non-native ones. These quality control processes involve proteins that sense folding-defective proteins and target them for destruction by a precisely controlled protein breakdown process known as ER-associated degradation (ERAD). The degradation of folding-defective glycoproteins is initiated in the lumen of the ER and the specific proteins that recognize the misfolded glycoproteins and determine their fate are unknown. We hypothesize that protein complexes consisting of alpha-mannosidases and thioredoxin-fold containing proteins are key factors in this process. The role of these complexes will be investigated using genetic, biochemical and cell biology approaches. The project will help to better understand protein quality control mechanisms in plants, which, in the long run, will lead to new strategies to improve plant fitness under constantly changing environmental conditions.

Wider research context Programmed cell death 1 receptor (PD-1) and its ligand programmed cell death ligand 1 (PD-L1) are immune checkpoint proteins that regulate the immune system. Under physiological conditions their interaction results in T-cell immune suppression, but cancer cells can hijack this pathway to escape immune detection by expressing PD-L1. Immunotherapies targeting the PD-1/PD-L1 axis represent a major breakthrough in cancer treatment. Several PD-1 and PD-L1 antibodies are FDA approved. However, their efficacy remains variable and weakly predictable, and thus attempts for the development of non-mAb PD-1/PD-L1 inhibitors are being pursued. PD-1 and PD-L1 are highly glycosylated proteins but the role of specific glycans in their interaction is poorly understood. Studies on the impact of glycosylation have focused on experimental studies using glyco-(null) mutants. Also, there are no reports on the impact of glycosylation on antibodies whose mode of action is receptor blockade and should not require ADCC or CDC activity. Clearly, there is a knowledge gap between our current understanding of immune regulation and how it can be manipulated to enhance the clinical efficacy of immune checkpoint blockade therapies in cancer. Objectives and methods The proposed project aims to take advantage of the transient expression system in N. benthamiana and its suitability for glyco-engineering to produce immune checkpoint proteins and antibodies with defined and homogenous human-like glycans. These will be used to determine the functional impact of glycans in the PD-1/PD-L1 interaction. In addition, glyco-engineered plant-derived proteins will be assessed for their binding affinity and effector functions in vitro. We expect to be able to identify glycan-optimized proteins that can act as decoy/trap molecules to inhibit the binding of PD-L1 in tumor cells to PD-1 in T-cells. Level of originality An in-depth analysis of the role of glycosylation in PD-L1/PD-1 interactions is not yet available. Studies on the functional activity of glyco-optimized proteins are likely to provide major insights into glycan-dependent interactions that will help guide therapeutic applications in cancer treatment. This research represents a promising new and fast way of producing and validating therapeutic recombinant proteins that can cut the time it takes to achieve significant clinical and experimental advances in cancer immunotherapy. Primary researchers involved Alexandra Castilho, with 27 years of experience in molecular genetics and cell biology and 18 in the field of plant N-glycosylation will be responsible for the execution of the project in close collaboration with experts in the fields of glyco-proteomics (Johannes Stadlmann); plant molecular farming (Lukas Mach and Waranyoo Phoolcharoen), immunology (Peter Steinberger) and glycosylation in cancer therapy (Celso Reis).

The plant-specific family of arabinogalactan proteins (AGPs) is implicated with a multitude of biological functions and their O-glycan might be crucial for either ligand interactions or for crude biophysical or structural protein properties. In this research proposal, however, I propose a role of O-glycosylation of the AGP type for protein fate by introducing the concept of an O-glycosylation checkpoint. I discuss evidence for the importance of O-glycosylation for protein fate in all eukaryotes and specifically describe the case of the moderately O-glycosylated FASCICLIN-LIKE ARABINOGALACTAN PROTEIN 4 (FLA4) from Arabidopsis thaliana. FLA4 abundance and localization strongly depends on its O-glycosylation. With a set of hydroxyproline-specific galactosyl transferases FLA4 acts in a linear genetic pathway necessary for normal root growth, salt tolerance and seed coat structure. Using FLA4 as genetic paradigm for a functional O-glycoprotein, I suggest hypothetical models of where and how O-glycosylation might influence the fate of plant proteins. I propose experimental approaches to elucidate the genetic and molecular mechanisms that determine the fate of FLA4 in dependence of its O-glycosylation status. Precise definition of proline hydroxylation and -glycosylation as well as molecular identification of crucial modification sites on FLA4, the cell biological elucidation of the involved organelles and the investigation of the degradation mechanism will comprise the first example for an endogenous plant protein that is controlled by its O-glycosylation state. Forward genetic isolation and next generation sequencing-based identification of suppressors of O-glycan dependent control of FLA4 abundance will provide novel genetic components of this process. A remarkable set of signalling proteins that have not previously been considered to be O-glycosylated, potentially also contain this modification. Therefore, it is likely that the O-glycan checkpoint acts on various regulatory pathways. The outcomes of this project will provide an important contribution to our fundamental knowledge of protein glycosylation and proteostasis and might thus contribute to improved stress tolerance of crop plants and the use of plants as factories