The Expression,Purification,and Functional Studies Of PDGF-D Prodomain

The platelate-derived growth factors(PDGFs)play important roles in embryonic development,vascular and tissue regenereation and homostasis.They regulate the proliferation of endothelial,fibroblasts,and smooth muscle cells,and promote the mitosis and chemostasis of these cells.These growth factors include: PDGF-A,PDGF-B,PDGF-C,and PDGF-D.Among them,the PDGF-A and PDGF-B are secreted as activated or pre-activated growth factors,while the PDGF-C and PDGF-D are secreted in a latent form,which needs to be activated in the local microenviroment.PDGF-B is the only ligand that is capable of binding to all known PDGF receptors.It not only promotes bone and stromal formation,but also leads to tumor angiogenesis,cancer cell proliferation and migration.PDGF-D has a different structure from PDGF-B.It contains a N-terminal,CUB-containing(Complement subcomponents C1r/C1 s,Urchin egf-like protein,and Bone marrow protein 1)prodomain,and a C-terminal growth factor domain.The prodomain of PDGF-D can sterically preventits growth factor domain from binding to the PDGF receptors.As such,PDGF-D is secreted in a latent form and is not able to trigger the downstream signaling without activation.Abnormal signaling of PDGF-B is closely related to the development and progress of various cancers,vascular and fibrosis diseases.Therefore,PDGF-B and its receptors had become important targets for therapeutic development.Based on our analysis of the structure and functions of PDGF-D,we proposed that its CUB-containing prodomain is critical for the latency and activation of PDGF-D.In addition,PDGF-D prodomain might have cross-inhibitory function on the growth factor activity of PDGF-B.To test these hypotheses,We constructed,expressed and purified the full-length PDGF-D,the recombinant proteins containing the PDGF-D CUB domain(Prodomain?Prodomain-Fc and GCN4-Prodomain),and the recombinant proteins containing the PDGF-D hinge region.We found that PDGF-D prodomain had the strongest inhibitory effect on the activity PDGF-B growth factor,and was able to inhibit PDGF-B-induced autopphosphorylation of PDGFR-? in a dose-dependent manner.This study could potentially be valuable for future study of the latency and activation of PDGF-D.It also provided a stepping stone in the development of PDGF-B targeted therapeutics.