Nicotiana prolyl-hydroxylase acting on proteins

The tobacco species Nicotiana benthamiana has emerged as practicable alternative to animal cells for the production of pharmaceutical proteins. Proline is an amino acid that in some proteins is modified by oxidation. The resulting hydroxyproline is a constituent of cartilage proteins such as collagens but it also occurs in plant proteins of the intercellular space. In plants it can be further modified by the sugar arabinose, which is not at all found in mammalian glycoproteins. This double modification is found in mugwort and ragweed pollen allergens, where it appears to play some role in binding of IgE antibodies from allergic individuals. With plants emerging as an expression system for drugs such as antibodies, the need is felt to avoid potentially harmful modifications of such drug proteins. The applicant and his collegues have been able to eliminate the most prominent unwanted modification, i.e. a certain protein-linked carbohydrate structure, from a tobacco line, which was already used to produce the renowned anti-Ebola antibody ZMAPP. Following this work, the present project will focus on the remaining problematic issue, i.e. proline oxidation. To this end, the enzymes responsible for proline oxidation shall be identified and Nicotiana benthamiana lines shall be generated, in which the relevant genes are suppressed. Proteins known to be targets of proline oxidation will then be produced in this line to verify the success of the strategy, which should finally lead to a production line for protein drugs of highest safety and tolerability. As the hydroxyprolines are the anchor point of the plant-specific addition of the sugar arabinose, the methods and materials used in this study lend themselves to also have a look at this somewhat elusive carbohydrate epitope and its role for allergic individuals.

Plants constitute a new production system for pharmaceutical proteins such as e.g. antibodies. For some proteins, plants can be an expression system superior to bacteria, yeasts or animal cells with regard to costs and/or product quality. However, some posttranslational modifications of the proteins are done in a different way as in humans. With regard to asparagine-linked carbohydrates this problem has been largely solved in the tobacco species frequently used for these purposes, i.e. Nicotiana benthamiana. A sore point, however, is the oxidation of proline at sites that are not modified in this way in mammals. Worse, once oxidized, arabinoses can be attached to these hydroxyproline residues. Such modification is unwanted. For a specialized expression system derived from moss, it was found that only one enzyme is responsible for proline oxidation. The now finalized project targeted for the first time the prolyl oxidases of vascular plants, in particular those of the above mentioned tobacco species. Erythropoietin (EPO) and the antibody class IgA1 - responsible for defence against respiratory pathogens (e.g. SARS-CoV 2) - were chosen as model proteins that were expressed in tobacco lines in which the one or the other prolyl oxidase gene was suppressed. The chosen combinations of suppression of gene expression showed promising but not yet sufficient results. On the basis of results of the now finalized project and with the help of the Crisp-Cas technology this shall be possible in an already running follow up project.