Nanobody-heavy chain (VHH-Fc) antibody formats have the potential to immunomodulate even highly accumulating proteins and provide a valuable tool to experimentally modulate the subcellular distribution of seed storage proteins. Recombinant antibodies often obtain high accumulation levels in plants, and thus, besides being the actual end-product, antibodies targeting endogenous host proteins can be used to interfere with the localization and functioning of their corresponding antigens. Here, we compared the effect of a seed-expressed nanobody-heavy chain (VHH-Fc) antibody against the highly abundant Arabidopsis thaliana globulin seed storage protein cruciferin with that of a VHH-Fc antibody without endogenous target. Both antibodies reached high accumulation levels of around 10% of total soluble protein, but strikingly, another significant part was present in the insoluble protein fraction and was recovered only after extraction under denaturing conditions. In seeds containing the anti-cruciferin antibodies but not the antibody without endogenous target, the amount of soluble, processed globulin subunits was severely reduced and a major part of the cruciferin molecules was found as precursor in the insoluble fraction. Moreover, in these seeds, aberrant vacuolar phenotypes were observed that were different from the effects caused by the depletion of globulins in knock-out seeds. Remarkably, the seeds with strongly reduced globulin amounts are fully viable and germinate with frequencies similar to wild type, illustrating how flexible seeds can retrieve amino acids from the stored proteins to start germination.

中文翻译：

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种子产生的抗球蛋白 VHH-Fc 抗体回收不溶性部分中的球蛋白前体并调节拟南芥种子亚细胞形态。

纳米抗体重链 (VHH-Fc) 抗体形式具有免疫调节甚至高度积累的蛋白质的潜力，并为通过实验调节种子储存蛋白的亚细胞分布提供了一种有价值的工具。重组抗体通常在植物中获得高积累水平，因此，除了作为实际的最终产物外，靶向内源宿主蛋白的抗体还可用于干扰其相应抗原的定位和功能。在这里，我们比较了种子表达的纳米抗体重链 (VHH-Fc) 抗体对高丰度拟南芥球蛋白种子储存蛋白十字花科蛋白的作用与没有内源性靶标的 VHH-Fc 抗体的作用。两种抗体都达到了总可溶性蛋白约 10% 的高积累水平，但引人注目的是，另一重要部分存在于不溶性蛋白质部分中，并且仅在变性条件下提取后才被回收。在含有抗十字花科植物素抗体但不含无内源性靶标的抗体的种子中，可溶性、加工球蛋白亚基的量严重减少，并且发现十字花科素分子的主要部分是不溶性部分中的前体。此外，在这些种子中，观察到异常的空泡表型，这与敲除种子中球蛋白耗尽所引起的影响不同。值得注意的是，球蛋白含量大大降低的种子完全有活力，并且以与野生型相似的频率发芽，说明灵活的种子如何从储存的蛋白质中提取氨基酸以开始发芽。