Combining SPR with atomic-force microscopy enables single-molecule insights into activation and suppression of the complement cascade

This work was supported by Leverhulme Trust Grant RPG-2015-109.Activation and suppression of the complement system compete on every serum-exposed surface, host or foreign. Potentially harmful outcomes of this competition depend on surface molecules through mechanisms that remain incompletely understood. Combining surface plasmon resonance (SPR) with atomic force microscopy (AFM), here we studied two complement system proteins at the single-molecule level: C3b, the proteolytically activated form of C3, and factor H (FH), the surface-sensing C3b-binding complement regulator. We used SPR to monitor complement initiation occurring through a positive-feedback loop wherein surface-deposited C3b participates in convertases that cleave C3, thereby depositing more C3b. Over multiple cycles of flowing factor B, factor D, and C3 over the SPR chip, we amplified C3b from ∼20 to ∼220 molecules·μm−2. AFM revealed C3b clusters of up to 20 molecules and solitary C3b molecules deposited up to 200 nm away from the clusters. A force of 0.17 ± 0.02 nanonewtons was needed to pull a single FH molecule, anchored to the AFM probe, from its complex with surface-attached C3b. The extent to which FH molecules stretched before detachment varied widely among complexes. Performing force-distance measurements with FH(D1119G), a variant lacking one of the C3b-binding sites and causing atypical hemolytic uremic syndrome, we found that it detached more uniformly and easily. In further SPR experiments, KD values between FH and C3b on a custom-made chip surface were 5-fold tighter than on commercial chips and similar to those on erythrocytes. These results suggest that the chemistry at the surface on which FH acts drives conformational adjustments that are functionally critical.Publisher PDFPeer reviewe

Complement C3a Enhances the Repair Potential of Human Mesenchymal Stem Cells

Abstract Abstract 1324 Mesenchymal stem cells (MSCs) contribute to the regulatory network in the bone marrow by generating the cellular compartment of the hematopoietic microenvironment including osteoblasts and stromal fibroblasts, and by producing a variety of trophic and growth factors. Because of these properties, MSCs are starting to find clinical application in a variety of pathological conditions including hematological disorders. However, the full regenerative potential of MSCs has not been realized due to poor tissue homing and limited cell survival following transplantation. One factor that has been largely ignored is that the local microenvironment, in which MSCs are expected to contribute to tissue regeneration, will provide an inflammatory milieu that is thought to be hostile to cell survival. Complement activation is one of the conditions that will be encountered by transplanted MSC in any area of tissue injury including post-chemotherapy or post-irradiation bone marrow. We therefore asked what the consequences of the complement activation products C3a and C5a are on MSCs. We found that complement fragment C3a causes activation of the ERK1/2 cascade in human MSCs, which lasts for several hours. C5a causes a similar, but less pronounced response. This is associated with translocation of ERK1/2 to the nucleus and results in transcriptional activation. Among the genes that are up-regulated following stimulation with C3a and, to a lesser degree, C5a are growth factors VEGF, bFGF, CXCL8/IL-8, IL-6, thrombopoeitin as well as TGF-beta1. Several of these factors are known to support hematopoiesis. C3a also caused transient activation of the Rho cascade in a pertussis toxin/Gi-protein-dependent fashion. Inhibition of the Rho cascade with the Rho kinase inhibitor Y27632 did not inhibit overall ERK1/2 phosphorylation, but prevented translocation of phospho-ERK1/2 and of the C3aR itself to the nucleus. This nuclear translocation of ERK1/2 was physiologically relevant, because inhibition of either the ERK1/2 cascade or Rho kinase, inhibited important functions of C3a on MSCs. This included the production of the afore-mentioned growth factors, C3a-dependent MSC migration and phosphorylation of the nuclear ERK target, Elk. Thus, activation of the Rho kinase pathway provided the means to regulate the spatial distribution of phosphorylated ERK1/2, and such signaling compartmentalization is a major mechanism by which non-specific ERK1/2 phosphorylation induced by different stimuli and in various cellular contexts can achieve specific outcomes. A similar role for Rho kinase in nuclear translocation of phospho-ERK1/2 has been reported previously in a variety of systems including cells stimulated with SDF-1/CXCL12. The exact molecular mechanism involved in all these instances is still not understood, but since the nuclear translocation of both phospho-ERK1/2 and of the C3aR was prevented in the presence of inhibition of the Rho cascade, it seems to be a general inhibitory effect on nuclear import likely related to cytoskeletal function. This effect of Rho kinase inhibition was specific for the effect of C3a, as C5a-induced chemotaxis was not influenced by Rho kinase inhibition, although it was largely ERK1/2 dependent. Our results indicate that physiologically important responses of MSCs to C3a, including cell migration and the production of growth factors depend on activation of the Rho kinase pathway and the ERK1/2 MAPK cascade. In addition to the ERK1/2 cascade, C3a and C5a also caused activation of the PI-3K/Akt pathway. Since both these signaling pathways protect from apoptosis, cell survival was determined in MSCs pre-exposed to C3a or C5a followed by exposure to oxidative injury. Under these conditions both C3a and C5a protected MSCs from apoptosis. In summary, C3a and C5a have three different effects: they are chemotactic factors for MSCs, they induce the production of various cytokines that support hematopoiesis, and they increase the MSC survival. These C3a and C5a-induced mechanisms will increase the therapeutic potential of MSCs, when MSCs are transplanted alone or given in conjuncture with hematopoietic stem cell transplantation. It furthermore indicates that complement activation initiates reparative rather than deleterious effects in MSCs. Disclosures: No relevant conflicts of interest to declare. </jats:sec