The impact of ER morphology on the assembly and interactions of sIgA in the endomembrane system of plants

Background.

In recombinant protein production, the efficiency of correct subunit assembly is often a limiting factor compromising the yield of functional product. Secretory IgAs, for example, pose a significant challenge for recombinant production in all heterologous systems, and even in mammalian cells they often require additional technological solutions, such as reassociation in vitro. Plant-based production systems, in particular those based on Nicotiana species, represented the first recombinant approach for the expression of secretory antibodies and demonstrated that the plant cellular machinery is well suited for the expression and assembly of sIgAs, but the overall yield and in particular the proportion of fully assembled protein still vary. Expanding the physical dimensions of the ER generally supports a high protein production rate and helps to alleviate the ER stress, and it has been reported that cells with artificially expanded ER show enhanced biosynthetic performance. We have therefore used CRISPR/Cas-mediated gene editing to artificially expand the ER network in N. benthamiana by increasing the biosynthesis of phosphatidylcholine (PC), which is the main building block of its constituent membranes. This resulted in a higher yield of fully assembled and functional SIgA in the mutant plant lines.

Hypotheses.

Since the retention time of the antibody was increased in the endomembrane system of the mutant plants, we hypothesize that the luminal volume and morphology of the endoplasmic reticulum influence the interactions between the antibody components and their interactions with ER-resident host factors.

Methods.

We will use MS-based identification to determine the interacting factors of the host cell machinery and draw a comparison between wt plants and existing mutants containing artificially expanded ER sheets via enhanced phospholipid synthesis. In addition, we will generate and test plants with a higher proportion of tubular ER structures via transient or stable overexpression of appropriate reticulons. Morphological changes of the endomembrane system will be monitored by 2D and 3D electron microscopy. Fluorescent labeling and imaging methods will be used to elucidate the temporal and spatial interaction patterns of the individual antibody components with each other and with ER-resident host factors. Finally, we will investigate the simultaneous overexpression of selected host proteins to equip the enlarged ER for optimal function.

Outcome.

We will develop a mechanistic understanding of the relationship between ER morphology and sIgA assembly. Identifying the relevant host factors will allow us to formulate novel and synergistic strategies for host cell engineering to optimize the production, assembly and modification of functional complex proteins.

For further reading please refer to Göritzer, K., Melnik, S., Schwestka, J., Arcalis, E., Drapal, M., Fraser, P., Ma, J.K.-C. and Stoger, E. (2025), Enhancing quality and yield of recombinant secretory IgA antibodies in Nicotiana benthamiana by endoplasmic reticulum engineering. Plant Biotechnol. J. https://doi.org/10.1111/pbi.14576