Montero-Morales Laura, Maresch Daniel, Castilho Alexandra, Turupcu Aysegül, Ilieva Kristina M, Crescioli Silvia, Karagiannis Sophia N, Lupinek Christian, Oostenbrink Chris, Altmann Friedrich, Steinkellner Herta
Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Vienna, Austria.
Department of Chemistry, University of Natural Resources and Life Sciences, Vienna, Austria.
J Proteomics. 2017 May 24;161:81-87. doi: 10.1016/j.jprot.2017.04.002. Epub 2017 Apr 8.
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.
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.
生物技术领域对人IgE抗体的兴趣日益浓厚，这就需要先进的系统来实现其正确表达。然而，由于该分子的复杂性，尤其是其多样的N-糖基化模式，这仍然是一个挑战。在此，我们展示了重组IgE在野生型和糖基工程改造的本氏烟草植物中的表达以及深入的N-糖基化分析。质谱分析表明，植物IgE的位点占据率范围从糖基化位点6（GS6）未占据到GS1和GS2的100%占据。与源自人细胞的IgE类似，植物来源的IgE在GS1 - 5处携带复杂型N-聚糖，在GS7处携带寡甘露糖结构。计算模型表明，聚糖的空间位置（或取向）可能会影响相邻糖基化位点的加工或位点占据。在糖基工程改造的植物和野生型植物中表达的IgE分别以高度均一性携带GnGn和植物典型的GnGnXF结构。这与源自HEK细胞的IgE的聚糖多样性形成对比，后者携带至少20种不同的糖型。重要的是，IgE糖基工程能够控制其糖基化，这在使用成熟的表达系统时是迄今未满足的需求。这有助于阐明可能的碳水化合物依赖性IgE功能。
重组蛋白的靶向糖基化可能在治疗应用中具有优势。尽管生物技术领域对IgE抗体的兴趣日益增加，但关于糖基化对这类抗体的了解和影响却很少。通过对重组IgE进行糖基工程改造的能力，我们朝着生成具有其他靶向N-聚糖的IgE迈出了重要一步。这将有助于进行详细的结构-功能研究，并可能导致产生具有优化活性的IgE。
