CCN2/CTGF binds the small leucine rich proteoglycan protein Tsukushi

Abstract

Extracellular molecules coordinate the multiple signaling pathways spatiotemporally to exchange information between cells during development. Understanding the regulation of these signal molecule-dependent pathways elucidates the mechanism of intercellular crosstalks. CCN2/CTGF is one of the CCN family members that binds BMP2, fibronectin, aggrecan, FGFR2 - regulating cartilage and bone formation, angiogenesis, wound repair etc. Tsukushi (TSK), which belongs to the Small Leucine-Rich Proteoglycan (SLRP) family, binds nodal/Vg1/TGF-β1, BMP4/chordin, Delta, FGF8, Frizzled4, and is involved in the early body formation, bone growth, wound healing, retinal stem cell regulation etc. These two secreted molecules are expressed in similar tissues and involved in several biological events by functioning as extracellular signaling modulators. Here, we examine the molecular interaction between CCN2 and TSK biochemically. Co-precipitation assay and Surface Plasmon Resonance measurement showed their direct binding with the Kd value 15.3 nM. Further, the Solid-phase Binding Assay indicated that TSK binds to IGFBP and CT domains of CCN2. Our data suggest that CCN2 and TSK exert their function together in the body formation.

Keywords: CCN2/CTGF; SLRP; Soluble molecule; Tsukushi; Vertebrate development.

Fig. 1

Direct interaction between CCN2 and TSK. a Western blot analysis of M-TSK-V5-His and CCN2-Flag-HA. b Co-precipitation of M-TSK-V5-His and CCN2-Flag-HA. After precipitation M-TSK-V5-His with nickel chelating resins, bound CCN2 was detected by immunoblotting with anti-HA antibody

Fig. 2

The binding of rCCN2 and rTSK was analyzed with SPR. The rCCN2 was coated on CM5 chip and serial dilutions of rTSK (46.875, 93.75, 187.5, 375 and 750 nM) were injected in ascending order of concentrations at 123, 246, 367, 505 and 628 s respectively. The Kd value was calculated from sensorgrams as described in Materials and methods. The data are representative of 2 independent experiments

Fig. 3

The bindings of FLAG-tagged rTSK to four domains of CCN2 (IGFBP, VWC, TSP1, or CT) or none were measured with solid-phase binding assay by using anti-FLAG mouse Ab and anti-mouse IgG-HRP as described in Materials and methods. The absorbance values at 450 nm indicates the amount of HRP bound to wells coated each domain of CCN2 or none. Statistical analysis was performed by performing Dunnett’s t-test and the One-way ANOVA test, *p < 0.001, as compared with the values for wells with 0 μg/ml rTSK. Data were expressed as the means of three independent wells ± standard deviation (SD)