The thrombospondin module 1 domain of the matricellular protein CCN3 shows an atypical disulfide pattern and incomplete CWR layers

Abstract

The members of the CCN (Cyr61/CTGF/Nov) family are a group of matricellular regulatory proteins that are essential to a wide range of functional pathways in cell signalling. Through interacting with extracellular matrix components and growth factors via one of their four domains, the CCN proteins are involved in critical biological processes such as angiogenesis, cell proliferation, bone development, fibrogenesis and tumorigenesis. Here, the crystal structure of the thrombospondin module 1 (TSP1) domain of CCN3 (previously known as Nov) is presented, which shares a similar three-stranded fold with the thrombospondin type 1 repeats of thrombospondin-1 and spondin-1, but with variations in the disulfide connectivity. Moreover, the CCN3 TSP1 domain lacks the typical -stacked ladder of charged and aromatic residues on one side of the domain that is seen in other TSP1 domains. Using conservation analysis among orthologous domains, it is shown that a charged cluster in the centre of the domain is the most conserved site and this cluster is predicted to be a potential functional epitope for heparan sulfate binding. This variant TSP1 domain has also been used to revise the sequence determinants of TSP1 domains and to derive improved Pfam sequence profiles for the identification of novel TSP1 domains in more than 10 000 proteins across diverse phyla. research papers Acta Cryst. (2020). D76, 124-134 Xu et al. Thrombospondin module 1 domain of CCN3 125

Multifunctional regulatory protein connective tissue growth factor (CTGF): A potential therapeutic target for diverse diseases

“…It contains both ‘CSXXCG’ sequence and six cysteines. In TSP1 domain, Trp206, Ser218, Arg220, Gln233 and Arg235 are 100% conserved across the CCN family proteins 43 , 44 . It was reported that TSP1 domain of CTGF could bind with exon 7-coded region of VEGF165 and negatively regulate the angiogenic activity of vascular endothelial growth factor (VEGF).…”

The carboxyl-terminal TSP1-homology domain is the biologically active effector peptide of matricellular protein CCN5 that counteracts profibrotic CCN2

“…Briefly, a fusion protein with signal peptide for secretion at the amino-terminal end and human albumin appended at the carboxyl-terminal end of the TSP1 domain (Alb-CCN5(III)) was expressed in CHO cells and purified from the cell culture media by sequential affinity chromatography and size-exclusion chromatography. Based on the extraordinary high degree of structural conservation of TSP1 homology domains among CCN proteins and report of the crystal structure of the TSP1 domain of CCN3 ( 8 ), we also made recombinant fusion protein of the TSP1 domain of CCN3 (Alb-CCN3(III)) configured similar to Alb-CCN5(III). As shown by the concentration-effect studies in Figure 1 B , Alb-CCN5(III) and Alb-CCN3(III) reduced phospho-AKT (Ser473) levels in A549 cells with similar efficacy and potency (IC 50 15.3 μg/ml and 6.8 μg/ml with 95% Confidence Intervals: 2.8–81.8 μg/ml and 4.2–10.8 μg/ml for Alb-CCN5(III) and Alb-CCN3(III), respectively).…”

“…TSP1 domains have been reported to exert functions through binding of many different membrane proteins or through binding of other proteins in the extracellular matrix. Among documented binding interactions are integrins, CD36, latent TGF-β, VEGF (vascular endothelial growth factor), collagen, fibronectin, and heparin sulfate–containing proteoglycans ( 8 , 48 , 49 , 50 , 51 , 52 , 53 ) (for review, see refs ( 54 , 55 )). As to CCN proteins, the TSP1 domain of CCN2 has been reported to bind VEGF 165 and sequester VEGF 165 from its receptors ( 52 ).…”

“…The two latter domains are separated from the two former amino-terminal domains by an unstructured “hinge region”, reported to be sensitive to various endopeptidases, including several members of the matrix metalloproteinase family (MMPs) ( 6 ). The crystal structure of the vWC and TSP1 domains of CCN3 have been reported, providing some structural insights with implications for all CCN family proteins ( 7 , 8 ). However, the overall globular structure is still missing.…”

“…Thus, we investigated in parallel the signaling and biological actions of these two domains. Following the recent report of the crystal structure of the TSP1 domain of CCN3 ( 8 ), functional differences among these domains might then be related to specific structural dissimilarities. The recombinant TSP1 domains were expressed as fusion proteins with various fusion partners in order to improve expression, secretion, solubility, and stability.…”