Recombinant plant-derived human IgE glycoproteomics.

The increasing biotechnological interest in human IgE antibodies demands advanced systems which allow their proper expression. However, this is still a challenge due to the complexity of the molecule, particularly regarding the diverse N-glycosylation pattern. Here, we present the expression of recombinant IgE in wild type and glycan-engineered Nicotiana benthamiana plants and in-depth N-glycosylation analyses. Mass spectrometric profiling revealed that plant IgE has a site occupancy rate that ranges from non-occupied at glycosite 6 (GS6) to 100% occupancy at GS1 and 2. Similarly to human cell-derived IgE, plant versions carry complex N-glycans at GS1-5 and oligomannosidic structures at GS7. Computational modelling suggests that spatial position (or orientation) of glycans can impair processing or site occupancy on adjacent glycosites. IgE expressed in glycoengineered and wild type plants carry, respectively, GnGn and plant-typical GnGnXF structures at large homogeneity. This contrasts with the glycan diversity of HEK cell-derived IgE, carrying at least 20 different glycoforms. Importantly, IgE glycoengineering allows the control of its glycosylation, a so far unmet need when using well-established expression systems. This enables the elucidation of possible carbohydrate-dependent IgE functions.

SIGNIFICANCE: Targeted glycosylation of recombinant proteins may provide an advantage in therapeutic applications. Despite increasing biotechnological interest in IgE antibodies, knowledge and impact of glycosylation on this antibody class are scarce. With the ability to glyco-engineer recombinant IgE, we provide an important step towards the generation of IgE with other targeted N-glycans. This will facilitate detailed structure-function studies and may lead to the production of IgE with optimized activities.