Domain-specific CCN3 antibodies as unique tools for structural and functional studies

Abstract

CCN3 is a member of the CCN family of cell growth and differentiation regulators that play key roles during embryonic development, and are associated with severe human pathologies. The level of CCN genes' expression is of prognostic value in several types of tumors. In the present manuscript, we report the isolation and characterization of a new set of antibodies targeted against each individual module of the human CCN3 protein. The need for module-specific antibodies stemmed from recent reports indicating that the expression of truncated CCN variant proteins was associated with development of cancers. Each of the four CCN3 modules were expressed as GST fusion proteins and used for rabbits immunization. Polyclonal IgGs purified by two rounds of affinitychromatography specifically detected both the individual CCN3 domains and the full length CCN3 protein expressed in mammalian cell lines and tissues, as well as recombinant full length and truncated CCN3 proteins. The purified module-specific antibodies were successfully used for Western blotting, immunoprecipitation, immunofluorescence and immunocytochemistry. These antibodies permitted the detection of CCN3 proteins under native and denaturing conditions, and confirmed the sublocalisation of CCN3 proteins in the extracellular compartment, at the cell membrane, in the cytoplasm and in the nucleus of positive cells. Immunocytochemistry and Western blotting studies performed with the module-specific antibodies identified truncated CCN3 proteins in kidney tumor samples. The detection of these rearranged variants provides clues for their involvement in tumorigenesis. Therefore, these antibodies constitute unique tools for the identification of truncated CCN3 proteins in human tissues and may be of great interest in molecular medicine.

CCN3/Nephroblastoma Overexpressed Matricellular Protein Regulates Integrin Expression, Adhesion, and Dissemination in Melanoma

“…A 32-kDa amino-truncated form is also found both in secreted and in the nucleus of different cell types. This short CCN3 isoform was described to lack the N-terminal domain, as determined by reactivity with the K19M antibody recognizing the C terminal (11) and confirmed recently with antibodies NH2 and NH5 specific for the N-terminal or the C-terminal part of CCN3, respectively (12). Protein sequence analysis (13) and lack of evidence for CCN3 alternative splicing suggest that the 32-kDa protein originates from posttranslational processing of the full-length protein (10).…”

“…1A, right). However, the absence of the full-length protein in the culture medium, as revealed using either antibody K19M or the more sensitive NH5 antibody (12), suggests that mechanisms other than posttranslational proteolysis of the secreted protein are also at play. Different patterns of CCN3 protein expression have also been detected in chronic myeloid leukemia (28) and in other tumor types (Perbal, unpublished results).…”

“…Proteins obtained from melanoma cells were separated on 4% to 12% bis-Tris precast gels (Invitrogen), transferred to nitrocellulose membranes (Amersham), and blotted with anti-CCN3 rabbit polyclonal antibodies K19M (11) and NH5 specific for the C-terminal CCN3 domain (12), rabbit anti-actin (Sigma-Aldrich), and monoclonal antibodies (mAb) against fibronectin, NBS1, and MHC class I W6/32 (BD Transduction). Reactive proteins were visualized using horseradish peroxidase (HRP)-conjugated secondary antibodies (Amersham) followed by enhanced chemiluminescence (GE Healthcare).…”

Matricellular Protein CCN3 (NOV) Regulates Actin Cytoskeleton Reorganization

“…Intracellular CCN3 variants have been previously detected in the cytoplasm and in the nucleus of some cancer cell lines (3,40,57,58). A smaller protein has been detected using a C-terminal specific but not an N-terminal specific antibody, and it is widely believed that the presence of intracellular CCN3 is either because of a proteolytic cleavage event or derived from an alternately spliced mRNA (3,27,58,59). Although we did detect a smaller protein at 25 kDa in the breast cell lysate but not in the culture medium using a C-terminal CCN3-specific antibody (Fig.…”

Regulatory role of CCN3 in melanoma cell interaction with the extracellular matrix

“…This 'short-form' originates from post-translational processing of the 'full-length' CCN3 protein and lacks both IGFBP and VWC domains. 6 The full-length secreted CCN3 acts for melanoma cells as an ECM protein itself and it mediates adhesion to other ECM proteins via integrin (ITG) receptors. 3 Clearly CCN3 modifies the adhesive properties of melanoma cells, in line with results previously obtained in Ewing's sarcoma by Benini and coworkers.…”