Recombinant expression, purification and functional impact of CTGF/CCN2 and NOV/CCN3 on liver fibrogenesis

Background: The CCN family of proteins, especially its prominent member, the Connective tissue growth factor (CTGF/CCN2/) has been identified as a possible biomarker for the diagnosis of fibrotic diseases [1]. As a downstream mediator of TGF-β1, it regulates expression and deposition of extracellular matrix proteins and promotes cell proliferation [2]. Another member of this family, the Nephroblastoma-overexpressed gene (NOV/CCN3), has growth-inhibiting properties. First reports further suggest that these two CCN proteins act opposite to each other [3]. Methods: We have established stable transfected HEK and Flp-In-293 clones and adenoviral vectors as productive sources for CTGF/CCN2 and NOV/CCN3 and established protocols to purify large quantities of these proteins. The biological integrity of purified proteins was tested in reporter and proliferation assays. The influence of transient overexpression for fibrosis-associated marker genes was analysed in culture-activated hepatic stellate cells and models of ongoing fibrogenesis. Results: The identity of purified proteins was proven by In-gel digest followed by ESI-MS/MS mass spectrometry. The total masses was determined by MALDI-TOF/TOF and biological activity was demonstrated using a Smad3-sensitive reporter gene and BrdU proliferation assay in EA.hy 926 cells. Most interestingly, the treatment with Peptid-N-(N-acetyl-β-glucosaminyl)-asparaginamidase and Endo-N-acetyl-β-glucosaminidase H further revealed that both CCN proteins are N-glycosylated. Conclusions: Both CCN proteins act differentially in regulating TGF-β signalling and oppositely influence their own expression. The opportunity to prepare large quantities of biological active CTGF/CCN2 and NOV/CCN3 proteins will now allow us to address key questions of these CCN proteins in regard to structure, function, post-translational modification and to delineate their impact on cellular signalling and initiation and progression of hepatic disease.