Chisholm Lauren O, Jeon Chae Kyung, Prell James S, Harms Michael J
Department of Chemistry & Biochemistry, University of Oregon, Eugene OR 97403.
Institute of Molecular Biology, University of Oregon, Eugene, OR 97403.
bioRxiv. 2024 Aug 14:2024.08.14.608001. doi: 10.1101/2024.08.14.608001.
S100A9 is a Damage Associated Molecular Pattern (DAMP) that activates the innate immune system via Toll-like receptor 4 (TLR4). Despite many years of study, the mechanism of activation remains unknown. To date, much of the biochemical characterization of S100A9 has been performed using recombinant S100A9 expressed in (S100A9). TLR4 is the canonical receptor for LPS, a molecule found in the outer membrane of , raising the possibility of artifacts due to LPS contamination. Here we report characterization of LPS-free recombinant S100A9 expressed in insect cells (S100A9). We show that S100A9 does not activate TLR4. This difference does not appear to be due to LPS contamination, protein misfolding, purification artifacts, or differences in phosphorylation. We show instead that S100A9 adopts an altered oligomeric state compared to S100A9. Disrupting oligomer formation with the disaggregase SlyD restores activity to S100A9. Our results also indicate that the oligomeric state of S100A9 is a major factor in its ability to activate TLR4 and that this can be altered in unexpected ways by the recombinant expression system used to produce the protein.
S100A9是一种损伤相关分子模式（DAMP），可通过Toll样受体4（TLR4）激活先天免疫系统。尽管经过多年研究，其激活机制仍不清楚。迄今为止，S100A9的许多生化特性表征都是使用在大肠杆菌中表达的重组S100A9（rS100A9）进行的。TLR4是脂多糖（LPS）的经典受体，脂多糖是一种存在于革兰氏阴性菌外膜中的分子，这增加了因LPS污染而产生假象的可能性。在这里，我们报告了在昆虫细胞中表达的无LPS重组S100A9（iS100A9）的特性表征。我们表明iS100A9不会激活TLR4。这种差异似乎不是由于LPS污染、蛋白质错误折叠、纯化假象或磷酸化差异造成的。相反，我们表明与rS100A9相比，iS100A9呈现出改变的寡聚状态。用解聚酶SlyD破坏寡聚体形成可恢复rS100A9的活性。我们的结果还表明，iS100A9的寡聚状态是其激活TLR4能力的一个主要因素，并且这可以通过用于生产该蛋白质的重组表达系统以意想不到的方式改变。
