The proteome of agroinfiltrated Nicotiana benthamiana is shaped by extensive protein processing
The proteome of agroinfiltrated Nicotiana benthamiana is shaped by extensive protein processing
复制标题
DOI：
10.1101/2023.11.02.565301
复制
发表时间：
2024-01
期刊：
影响因子：
--
通讯作者：
Kaijie Zheng;Joy C. Lyu;Emma L. Thomas;Mariana Schuster;N. Sanguankiattichai;Sabrina Ninck;F. Kaschani;Markus Kaiser;R. V. D. van der Hoorn
中科院分区：
文献类型：
--
作者：
Kaijie Zheng;Joy C. Lyu;Emma L. Thomas;Mariana Schuster;N. Sanguankiattichai;Sabrina Ninck;F. Kaschani;Markus Kaiser;R. V. D. van der Hoorn
蛋白水解酶的加工不可逆转地调节植物蛋白质的命运，阻碍植物中重组蛋白的生产，然而在农业渗透的本氏烟草中只有很少的加工事件被描述，它已经成为植物科学和分子药学中最受欢迎的瞬时蛋白质表达平台。在这里，我们使用凝胶内消化和质谱仪来监测5040种植物蛋白在蛋白凝胶中的迁移和地形。通过在凝胶切片上绘制多肽，我们生成了胃肠图，揭示了在蛋白质凝胶中检测到每种蛋白质的哪一部分。这些数据发现，60%的检测到的蛋白质具有比预测的相对分子质量低的蛋白质形式，这意味着广泛的蛋白质分解过程。例如，这项分析证实了大多数但不是所有蛋白酶的自抑制原结构域的蛋白水解性去除和降解，并揭示了果胶甲基酯酶和脂肪酶家族之间的不同处理。这项分析还揭示了糖苷酶的复杂加工，并发现胞外结构域脱落可能在一系列不同的受体样激酶中很常见。双标记候选蛋白的瞬时表达证实了体内的各种加工事件。这一广泛的蛋白质组数据可以进一步研究，并表明大多数植物蛋白质都是经过蛋白质水解性加工的，这意味着一种广泛的蛋白质分解机制塑造了农业渗透的本氏新月球藻的蛋白质组。
Processing by proteases irreversibly regulates the fate of plant proteins and hampers the production of recombinant protein in plants, yet only few processing events have been described in agroinfiltrated Nicotiana benthamiana, which has emerged as a favorite transient protein expression platform in plant science and molecular pharming. Here, we used in-gel digests and mass spectrometry to monitor the migration and topography of 5,040 plant proteins of agroinfiltrated N. benthamiana within a protein gel. By plotting the peptides over the gel slices, we generated peptographs that reveal where which part of each protein was detected within the protein gel. These data uncovered that 60% of the detected proteins have proteoforms that migrate at lower than predicted molecular weights, implicating extensive proteolytic processing. For instance, this analysis confirms the proteolytic removal and degradation of autoinhibitory prodomains of most but not all proteases, and revealed differential processing within pectinemethylesterase and lipase families. This analysis also uncovered intricate processing of glycosidases and uncovered that ectodomain shedding might be common for a diverse range of receptor-like kinases. Transient expression of double-tagged candidate proteins confirmed various processing events in vivo. This extensive proteomic dataset can be investigated further and demonstrates that most plant proteins are proteolytically processed and implicates an extensive proteolytic machinery shaping the proteome of agroinfiltrated N. benthamiana.