Chisholm, L. O. | Search bioRxiv

bioRxiv2024

Changing expression system alters oligomerization and proinflammatory activity of recombinant human S100A9

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 E. coli (S100A9ec). TLR4 is the canonical receptor for LPS, a molecule found in the outer membrane of E. coli, raising the possibility of artifacts due to LPS contamination. Here we report characterization of LPS-free recombinant S100A9 expressed in insect cells (S100A9in). We show that S100A9in 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 S100A9in adopts an altered oligomeric state compared to S100A9ec. Disrupting oligomer formation with the E. coli disaggregase SlyD restores activity to S100A9in. 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.

biochemistry↗

bioRxiv2024

S100A9 interacts with a dynamic region on CD14 to activate Toll-like receptor 4.

S100A9 is a Damage Associated Molecular Pattern (DAMP) that activates inflammatory pathways via Toll-like receptor 4 (TLR4). This activity plays important homeostatic roles in tissue repair, but can also contribute to inflammatory diseases. The mechanism of activation is unknown. Here, we follow up on a previous observation that the protein CD14 is an important co-receptor that enables S100A9 to activate TLR4. Using cell-based functional assays and a combination of mutations and pharmocological perturbations, we found that CD14 must be membrane bound to potentiate TLR4 activation by S100A9. Additionally, S100A9 is sensitive to inhibitors of pathways downstream of TLR4 internalization. Together, this suggests that S100A9 induces activity via CD14-dependent internalization of TLR4. We then used mutagenesis, structural modeling, and in vitro binding experiments to establish that S100A9 binds to CD14s N-terminus in a region that overlaps with, but is not identical to, the region where CD14 binds its canonical ligand, lipopolysaccharide (LPS). In molecular dynamics simulations, this region of the protein is dynamic, allowing it to reorganize to recognize both S100A9 (a soluble protein) and LPS (a small hydrophobic molecule). Our work is the first attempt at a molecular characterization of the S100A9/CD14 interaction, bringing us one step closer to unraveling the full mechanism by which S100A9 activates TLR4/MD-2.

biochemistry↗