Nodulin proteins are expressed in legume root cells after infection with rhizobia. Nodulins have been classified as early and late, reflecting the developmental time points of their expression. Leghemoglobin (LegH), which is a classical example of a late nodulin, sequesters oxygen inside the nodule to protect the nitrogenase from oxygen toxicity to sustain symbiotic nitrogen fixation (SNF). Post-translational modification and/or protein–protein interaction are known to regulate activity of proteins. To elucidate the role of post-translational modification of LegH on its oxygen sequestration activity, earlier we have shown that phosphorylation at its S45 imparts most structural disruption of the porphyrin binding pocket responsible for its oxygen binding. In the present report, in an attempt to characterize the protein(s) that may interact with LegH to regulate its activity, it is demonstrated that LegH interact in vitro with Nodulin 16 of Lotus japonicus (Nlj16), another late nodulin. These two interacting proteins resulted in a bigger sized particle which shows higher diffusion coefficient as measured by dynamic light scattering. Interestingly, it was also shown that in vitro oxygen sequestration by LegH is stimulated by this interaction. Furthermore, this interaction is validated by the fact that LegH and Nlj16 could be co-immunoprecipitated from the nodule lysate. Most importantly, fluorescent immunohistochemistry of post-infected nodule sections show perceivable co-localization of these two proteins in the nodule symbiosomes. Thus, this work is a foundation for further investigation on these two interacting late nodulins as one of the plausible regulations for the oxygen sequestration by LegH during SNF.

中文翻译：

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豆科血红蛋白对氧的螯合作用通过其与另一晚结节蛋白（日本莲的Nlj16）的相互作用而受到正调控。

根瘤菌感染后在豆类根细胞中表达结节蛋白。结节蛋白已分类为早期和晚期，反映了其表达的发育时间点。豆球蛋白（LegH）是晚期结节蛋白的经典例子，它在结节中隔离氧，以保护固氮酶免受氧中毒，从而维持共生固氮（SNF）。已知翻译后修饰和/或蛋白质-蛋白质相互作用可调节蛋白质的活性。为了阐明LegH的翻译后修饰对其氧螯合活性的作用，我们早些时候已经表明，在其S45处的磷酸化作用使卟啉结合口袋的大部分结构破坏，从而导致了其氧结合。在本报告中，莲花japonicus（Nlj16），另一种晚结节蛋白。这两种相互作用的蛋白质产生较大尺寸的颗粒，如通过动态光散射测量的，该颗粒显示出较高的扩散系数。有趣的是，还显示了通过这种相互作用刺激了LegH的体外氧隔离。此外，LegH和Nlj16可以从结节溶解产物中被免疫共沉淀这一事实证实了这种相互作用。最重要的是，感染后结节切片的荧光免疫组织化学显示这两个蛋白在结节共生体中可感知的共定位。因此，这项工作为进一步研究这两种相互作用的晚期结节蛋白奠定了基础，这是LegH在SNF期间螯合氧的合理规定之一。