Heparan sulfate dependent phase separation of CCL5 and its chemotactic activity

Secreted chemokines form concentration gradients in target tissues to control migratory directions and patterns of immune cells in response to inflammatory stimulation; however, how the gradients are formed is much debated. Heparan sulfate (HS) binds to chemokines and modulates their activities. In this study, we investigated the roles of HS in the gradient formation and chemoattractant activity of CCL5 that is known to bind to HS. CCL5 and heparin underwent liquid-liquid phase separation (LLPS) and formed gradient, which was confirmed using CCL5 immobilized on heparin-beads. The biological implication of HS in CCL5 gradient formation was established in CHO-K1 (wild type) and CHO-677 (lacking HS) cells by Transwell assay. The effect of HS on CCL5 chemoattractant activity was further proved by Transwell assay of human peripheral blood cells. Finally, peritoneal injection of the chemokines into mice showed reduced recruitment of inflammatory cells either by mutant CCL5 (lacking heparin binding sequence) or by addition of heparin to wild type CCL5. Our experimental data propose that co-phase separation of CCL5 with HS establishes a specific chemokine concentration gradient to trigger directional cell migration. The results warrant further investigation on other heparin binding chemokines and allows for a more elaborate insight into disease process and new treatment strategies.