Bouargalne Youssef, Guilbaud Florian, Macherel David, Delalande Olivier, Deleu Carole, Le Cahérec Françoise
IGEPP, INRAE, Institut Agro, Université Rennes, Rennes 35000, France.
IRHS, INRAE, Institut Agro, Université Angers, Angers 49000, France.
Plant Cell Physiol. 2023 May 15;64(5):536-548. doi: 10.1093/pcp/pcad016.
Class II water-soluble chlorophyll proteins (WSCPs) from Brassicaceae are non-photosynthetic proteins that bind with chlorophyll (Chl) and its derivatives. The physiological function of WSCPs is still unclear, but it is assumed to be involved in stress responses, which is likely related to their Chl-binding and protease inhibition (PI) activities. Yet, the dual function and simultaneous functionality of WSCPs must still be better understood. Here, the biochemical functions of Brassica napus drought-induced 22-kDa protein (BnD22), a major WSCP expressed in B. napus leaves, were investigated using recombinant hexahistidine-tagged protein. We showed that BnD22 inhibited cysteine proteases, such as papain, but not serine proteases. BnD22 was able to bind with Chla or Chlb to form tetrameric complexes. Unexpectedly, BnD22-Chl tetramer displays higher inhibition toward cysteine proteases, indicating (i) simultaneous Chl-binding and PI activities and (ii) Chl-dependent activation of PI activity of BnD22. Moreover, the photostability of BnD22-Chl tetramer was reduced upon binding with the protease. Using three-dimensional structural modeling and molecular docking, we revealed that Chl binding favors interaction between BnD22 and proteases. Despite its Chl-binding ability, the BnD22 was not detected in chloroplasts but rather in the endoplasmic reticulum and vacuole. In addition, the C-terminal extension peptide of BnD22, which cleaved off post-translationally in vivo, was not implicated in subcellular localization. Instead, it drastically promoted the expression, solubility and stability of the recombinant protein.
十字花科植物的II类水溶性叶绿素蛋白（WSCPs）是与叶绿素（Chl）及其衍生物结合的非光合蛋白。WSCPs的生理功能尚不清楚，但推测其参与应激反应，这可能与其叶绿素结合和蛋白酶抑制（PI）活性有关。然而，WSCPs的双重功能和同时发挥的功能仍需深入了解。在此，我们使用重组六聚组氨酸标签蛋白研究了甘蓝型油菜干旱诱导的22 kDa蛋白（BnD22）的生化功能，BnD22是甘蓝型油菜叶片中表达的一种主要WSCP。我们发现BnD22抑制半胱氨酸蛋白酶，如木瓜蛋白酶，但不抑制丝氨酸蛋白酶。BnD22能够与叶绿素a或叶绿素b结合形成四聚体复合物。出乎意料的是，BnD22-叶绿素四聚体对半胱氨酸蛋白酶的抑制作用更强，这表明（i）其同时具有叶绿素结合和PI活性，以及（ii）叶绿素依赖性激活BnD22的PI活性。此外，BnD22-叶绿素四聚体与蛋白酶结合后光稳定性降低。通过三维结构建模和分子对接，我们发现叶绿素结合有利于BnD22与蛋白酶之间的相互作用。尽管BnD22具有叶绿素结合能力，但在叶绿体中未检测到它，而是在内质网和液泡中检测到。此外，BnD22的C末端延伸肽在体内翻译后被切割掉，它与亚细胞定位无关。相反，它极大地促进了重组蛋白的表达、溶解性和稳定性。
